Methods and compositions for increasing toxin production

ABSTRACT

The invention provides methods and compositions (such as for example, culture media) for culturing  Clostridium difficile  and producing the  C. difficile  Toxins A and B.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the U.S. national stage filing under 35 U.S.C. §371of international application PCT/US2009/058268, filed Sep. 24, 2009,which claims benefit of Provisional Patent Application. 61/099,759,filed Sep. 24, 2008.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. Provisional PatentApplication No. 61/099,759, filed on Sep. 24, 2008, which isincorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

Clostridium difficile (C. difficile) Toxins A and B are responsible forC. difficile-associated disease (CDAD), which manifests itself asnosocomial diarrhea and pseudomembranous colitis (Kuijper et al.,Clinical Microbiology and Infection 12(Suppl. 6):2-18, 2006; Drudy etal., International Journal of Infectious Diseases 11(1):5-10, 2007;Warny et al., Lancet 366(9491):1079-1084, 2005; Dove et al., Infectionand Immunity 58(2):480-488, 1990; Barroso et al., Nucleic Acids Research18(13):4004, 1990).

Toxins A and B are encoded by two separate but closely linked (andhighly homologous) genes. Toxins A and B are produced simultaneously inC. difficile strain VPI 10463 (ATCC 43255), and the ratio of theproduced toxins is usually 3:1, respectively (Karlsson et al.,Microbiology 145:1683-1693, 1999). The toxins begin to be formed duringthe exponential growth phase, and are usually released from the bacteriabetween 36 and 72 hours of culture. Toxins present within the bacteriacan be released earlier by sonication or by use of a French pressurecell.

Treatment of the toxins with formaldehyde results in the correspondingToxoids A and B, which are completely inactivated and retain at leastpartial immunogenicity (Torres et al., Infection and Immunity63(12):4619-4627, 1995). It has been shown that vaccination employingboth toxoids is effective in hamsters, healthy adults, and patients withrecurrent CDAD (Torres et al., Infection and Immunity 63(12):4619-4627,1995; Kotloff et al., Infection and Immunity 69(2):988-995, 2001;Sougioultzis et al., Gastroenterology 128(3):764-770, 2005; Tones etal., Vaccine Research 5(3):149-162, 1996). Additionally, theadministration of both free and aluminum salt (adjuvant) bound toxoidsleads to appropriate immune responses (Torres et al., Vaccine Research5(3):149-162, 1996; Giannasca et al., Infection and Immunity67(2):527-538, 1999).

The administration of both toxoids simultaneously is more effective thanadministration of the individual proteins alone (Kim et al., Infectionand Immunity 55(12):2984-2992, 1987). A toxoid composition foundeffective in inducing protective immune responses against toxin A andtoxin B in patients with recurrent CDAD included both toxoids, at aratio of 1.5:1, A:B (Sougioultzis et al., Gastroenterology128(3):764-770, 2005).

Both the A and B toxoids are thus candidates for vaccine development.Greater production efficiency of Toxins A and B is desired to facilitatevaccine production.

SUMMARY OF THE INVENTION

In one aspect, the invention features a culture medium (e.g., forculturing a Clostridium difficile bacterium) at a pH of between 6.35 and7.45 (e.g., 6.5, 7.28, or between 6.35 and 6.65) including peptone(e.g., soy peptone), a yeast extract (e.g., Difco Bacto Yeast extract),a buffering agent (e.g., NaHCO₃), and a phosphate buffer (e.g., sodiumphosphate, dibasic and potassium phosphate, monobasic). This culturemedium can also include at least one additive (e.g., 2, 3, or moreadditives) selected from the group consisting of chromium trioxide,clindamycin, ascorbic acid, butyric acid, D(+)xylose, D-sorbitol,sucrose, and a combination of azaserine, adenosine, and biotin.

In another aspect, the invention features a bacterial culture includingClostridium difficile and culture medium including at least one additive(e.g., 2, 3, or more additives) selected from the group consisting ofchromium trioxide, clindamycin, ascorbic acid, butyric acid, D(+)xylose,D-sorbitol, sucrose, and a combination of azaserine, adenosine, andbiotin. This medium can also include peptone (e.g., soy peptone), yeastextract (e.g., Difco Bacto Yeast extract), sodium phosphate, dibasic,potassium phosphate, monobasic, and NaHCO₃, and the culture medium canbe at a pH of between 6.35 and 7.45 (e.g., 6.5, 7.28, or between 6.35and 6.65).

In another aspect, the invention features a method of culturingClostridium difficile including inoculating culture medium withClostridium difficile, with the medium including at least one additive(e.g., 2, 3, or more additives) selected from the group consisting ofchromium trioxide, clindamycin, ascorbic acid, butyric acid, D(+)xylose,D-sorbitol, sucrose, and a combination of azaserine, adenosine, andbiotin. This medium can also include peptone (e.g., soy peptone), yeastextract (e.g., Difco Bacto Yeast extract), sodium phosphate, dibasic,potassium phosphate, monobasic, and NaHCO₃, and the culture medium canbe at a pH of between 6.35 and 7.45 (7.28 or between 6.35 and 6.65).Preferably the culture medium is at a pH of 6.5.

In another aspect, the invention features a method for obtaining orpreparing one or more C. difficile toxins including by preparing anaqueous growth medium including soy peptone, inoculating the medium witha C. difficile bacterium (e.g., using an aqueous C. difficile culture),culturing the inoculated medium (e.g., at a pH of 6.5, 7.28, between6.35 and 7.45, or between 6.35 and 6.65)) under conditions whichfacilitate growth of bacterium and toxin production (e.g., at atemperature between 37° C. to 41° C.), and isolating the one or more C.difficile toxins from growth medium (e.g., by removing from the growthmedium viable C. difficile organisms and spores, separating the one ormore toxins from the growth media, and purifying the one or moretoxins). This culture medium can also include yeast extract, NaHCO₃,sodium phosphate, dibasic, potassium phosphate, monobasic, andD-sorbitol. This culturing can be carried out, e.g., under anaerobicconditions. The steps of inoculating the medium with a C. difficilebacterium (e.g., using an aqueous C. difficile culture) and culturingthe inoculated medium can be repeated more than once, with inoculationinto fresh growth medium with each repeat. This method can also includedetoxifying the isolated one or more C. difficile toxins to prepare oneor more toxoids (e.g., by reacting the one or more toxins by theaddition of formaldehyde).

In another aspect, the invention features a method of enhancing theproduction of Toxin B from a C. difficile culture by preparing anaqueous growth medium including soy peptone, inoculating the medium witha C. difficile bacterium, culturing the inoculated medium at 37° C. to41° C. and at a pH between pH 6.35 and pH 6.65 (e.g., at 37° C. and at apH of 6.5). The pH and/or temperature can be held constant or varyduring the culturing. The growth media can further include yeastextract, NaHCO₃; sodium phosphate, dibasic, potassium phosphate,monobasic, and D-sorbitol. Toxin B production can be enhanced relativeto Toxin A production, producing, e.g., ratios of Toxin A relative toToxin B of less than 3:1, 2:1, 1.5:1, or less.

In any of the foregoing aspects, yeast extract can be between 10 and 30g/L, the NaHCO₃ can be between 2 and 5 g/L; the sodium phosphate,dibasic can be between 1 and 10 g/L, and the potassium phosphate,monobasic can be between 1 and 10 g/L. The adenosine can be present at aconcentration of between 0.8 and 1.2 mM (e.g., 1 mM), the biotin at aconcentration of between 40 and 60 nM (e.g., 50 nM), and the azaserineat a concentration between 15 and 50 μM (e.g., 50 μM). The concentrationof D-sorbitol can be between 6 g/L and 20 g/L or between 8 g/L and 18g/L (e.g., 12 g/L). The chromium trioxide can be present at aconcentration of between 40 and 60 mg/L (e.g., 50 mg/L). The clindamycincan be present at a concentration between 0.4 and 0.6 mg/L (e.g., 0.5mg/L). The ascorbic acid can be present at a concentration between 2.5g/L and 10 g/L (e.g., 2.5 g/L and 10 g/L). The butyric acid can bepresent at a concentration between 30 mM and 60 mM (e.g., 30 mM and 60mM). The D(+)xylose can be at a concentration between 6 g/L and 10 g/L(e.g., 6 g/L).

The invention provides several advantages. For example, the media andthe methods of the invention allow increased production of Clostridiumdifficile toxins, which leads to increased efficiency and decreasedcosts in the production of toxin-based products such as vaccines. Otherfeatures and advantages of the invention will be apparent from thefollowing Detailed Description, the Drawings, and the Claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-G are graphs showing the amount of production of the indicatedtoxin in cultures containing the indicated additive at the indicatedconcentration.

FIG. 2A is an SDS PAGE gel showing the amount of Toxin A produced incells cultured with the indicated compounds at 12 hours.

FIG. 2B is an SDS PAGE gel showing the amount of Toxin A produced incells cultured with the indicated compounds at 24 hours.

FIG. 3A is an SDS PAGE gel showing the amount of Toxin A produced incells cultured with the indicated compounds at 12 hours.

FIG. 3B is an SDS PAGE gel showing the amount of Toxin A produced incells cultured with the indicated compounds at 24 hours.

FIG. 4A is an SDS PAGE gel showing the amount of Toxin A produced incells cultured with the indicated compounds at 12 hours. The lanes wereloaded with samples from cultures including the following compounds:Control; #1 Arginine (50 mM); #2 Cysteine (0.33 mM); #3 Cysteine (3.3mM); #4 Cysteine (33 mM); #5 Tyrosine (50 mg/L); #6 Ascorbic acid (2.5g/L); #7 Ascorbic acid (10 g/L); #8 Butyric acid (30 mM); #9 Butyricacid (60 mM).

FIG. 4B is an SDS PAGE gel showing the amount of Toxin A produced incells cultured with the indicated compounds at 24 hours. The lanes wereloaded with samples from cultures including the following compounds: #1Arginine (50 mM); #2 Cysteine (0.33 mM); #3 Cysteine (3.3 mM); #4Cysteine (33 mM); #5 Tyrosine (50 mg/L); #6 Ascorbic acid (2.5 g/L); #7Ascorbic acid (10 g/L); #8 Butyric acid (30 mM); #9 Butyric acid (60mM).

FIG. 5A is an SDS PAGE gel showing the amount of Toxin A produced incells cultured with the indicated compounds at 12 hours. The lanes wereloaded with samples from cultures including the following compounds: #1D(−)Fructose (6 g/L); #2-D(+)Galactose (6 g/L); #3 Mannose (6 g/L); #4Maltose Monohydrate (6 g/L); #5 Sucrose (6 g/L); #6α-Lactose (6 g/L); #7D(+)Xylose (6 g/L); #8 D-Sorbitol (6 g/L); #9 myo-Inositol (6 g/L).

FIG. 5B is an SDS PAGE gel showing the amount of Toxin A produced incells cultured with the indicated compounds at 24 hours. The lanes wereloaded with samples from cultures including the following compounds:#1-D(−)Fructose (6 g/L); #2 D(+)Galactose (6 g/L); #3 Mannose (6 g/L);#4 Maltose Monohydrate (6 g/L); #5 Sucrose (6 g/L); #6α-Lactose (6 g/L);#7 D(+)Xylose (6 g/L); #8 D-Sorbitol (6 g/L); #9 myo-Inositol (6 g/L).

FIG. 6A is a graph showing the amount of Toxin A produced (ng/ml) incultures subject to the indicated pH control.

FIG. 6B is a graph showing the amount of Toxin B produced (ng/ml) incultures subject to the indicated pH control.

FIG. 7A is a graph showing the amount of Toxin A produced (ng/ml) incultures subject to the indicated pH control.

FIG. 7B is a graph showing the amount of Toxin B produced (ng/ml) incultures subject to the indicated pH control.

FIG. 7C is a graph showing specific Toxin A productivity produced (ng/mlper OD unit) in the cultures subject to the indicated pH control.

FIG. 7D is a graph showing specific Toxin B productivity produced (ng/mlper OD unit) in the cultures subject to the indicated pH control.

FIG. 7E is a graph comparing the specific Toxin A and Toxin B produced(ng/ml) in cultures subject to the indicated pH control in Examples 6and 7.

FIG. 8A is a graph showing the amount of Toxin A produced (ng/ml) incultures subject to the indicated pH control and the indicated amount ofsodium bicarbonate.

FIG. 8B is a graph showing the amount of Toxin B produced (ng/ml) incultures subject to the indicated pH control and the indicated amount ofsodium bicarbonate.

FIG. 9A is a graph showing the amounts of Toxin A and B produced (ng/ml)in 18 hour cultures subject to the indicated pH control in comparison tocell growth (OD600).

FIG. 9B is a graph showing the amounts of Toxin A and B produced (ng/ml)in 22 hour cultures subject to the indicated pH control in comparison tocell growth (OD600).

FIG. 10A is a graph showing the amount of Toxin A produced (ng/ml) incultures subject to the indicated temperature.

FIG. 10B is a graph showing the amount of Toxin B produced (ng/ml) incultures subject to the indicated temperature.

FIG. 10C is a graph showing specific Toxin A productivity produced(ng/ml per OD unit) in the cultures subject to the indicatedtemperature.

FIG. 10D is a graph showing specific Toxin B productivity produced(ng/ml per OD unit) in the cultures subject to the indicatedtemperature.

FIG. 11 is a graph showing the amount of Toxin A and Toxin B produced(ng/ml) in comparison to cell growth (OD600) in cultures subject to theindicated pH condition and inoculated with the indicated % of inoculum.

DETAILED DESCRIPTION

In general, the invention features methods and compositions (such as forexample, culture media) for culturing C. difficile and producing the C.difficile Toxins A and B. These two toxins can be used individually orin combination, in the preparation of toxoids.

As discussed further below, the culturing of C. difficile in the mediaof the invention leads to enhanced Toxin A and Toxin B production.Similarly, as discussed further below, enhanced toxin production is seenby culturing C. difficile in accordance to the methods of the invention.

Basal Media

The compositions and methods of the invention feature the use of a basalmedium in conjunction with certain medium additives. In one example, thebasal medium is comprised of peptone (e.g., 20-40 g/L), yeast extract(e.g., 10-30 g/L), a phosphate buffer (such as for example, potassiumphosphate monobasic (e.g., 0.5-1.5 g/L) and sodium phosphate dibasic(e.g., 1-10 g/L)) and a buffering agent (such as for example, sodiumbicarbonate (e.g., 1-10 g/L)). The peptone used may be soy-based oranimal-derived (such as for example, tryptone).

In one example, the basal medium is SYS media. SYS medium contains theingredients listed in Table 1A at the indicated concentrations. Thebasal medium may be titrated to a pH of between 6.35 and 7.45 (forexample, 6.5, 7.28, or between 6.35 and 6.65). Exemplary ranges ofconcentrations for each of the indicated ingredients are also indicated.

TABLE 1A Grams Acceptable range Ingredient per liter of grams per literSoy peptone A3 30 20-40 (e.g., 25-35 and 29-31) Difco Bacto Yeastextracts 20 10-30 (e.g., 15-25 and 19-21) KH₂PO₄ 0.9 0.5-1.5 (e.g.,0.8-1.0) Na₂HPO₄ 5 1-10 (e.g., 2-8 and 4-5) NaHCO₃ 5 1-10 (e.g., 2-8 and4-5)

Table 1B sets forth an alternative basal media useful in thecompositions and methods of the invention, named TYS.

TABLE 1B Grams Acceptable range Ingredient per liter of grams per literDifco Bacto Tryptone 30 20-40 (e.g., 25-35 and 29-31) Difco Bacto Yeastextracts 20 10-30 (e.g., 15-25 and 19-21) KH₂PO₄ 0.9 0.5-1.5 (e.g.,0.8-1.0) Na₂HPO₄ 5 1-10 (e.g., 2-8 and 4-5) NaHCO₃ 5 1-10 (e.g., 2-8 and4-5)

In substitution of the Soy peptone A3 and the Difco Bacto Tryptone anypeptone (e.g., any soy peptone) can be utilized. Examples of soypeptones that can be used in the basal media (and their sources) includethe following:

Kerry Biosciences: HyPer 1510,

IPS: Hy-Soy Kosher, and

Becton Dickinson: BD Select Phytone UF

In substitution of the Difco Bacto Yeast Extract, any yeast extract canalso be used in the basal media. Examples of suitable yeast extracts(and their sources) are readily known to those skilled the art.

The suitability of a particular peptone or yeast extract for use in theinvention can be determined using the experimental methods describedherein. The invention also includes use of other bacterial growth media,in combination with the additives described below.

Additives

The invention also features the use of certain additives with a basalmedia (e.g., SYS media). Exemplary additives of the invention are setforth in Table 2, which also includes the exemplary concentration rangesfor the indicated additives, as well as a single exemplaryconcentration. Additives include:

Chromium trioxide (Chromium(VI) oxide CrO₃). Chromium trioxide is theacid anhydride of chromic acid. Chromium trioxide is a strong oxidant,highly toxic, corrosive, and carcinogenic compound.

Clindamycin (C₁₈H₃₃ClN₂O₅S). Clindamycin is a lincosamide antibiotic andis indicated for Clostridium difficile-associated diarrhea (the mostfrequent cause of pseudomembranous colitis). Clindamycin has abacteriostatic effect. It interferes with bacterial protein synthesis bybinding preferentially to the 50S subunit of the bacterial ribosome.

Azaserine (C₅H₇N₃O₄). Azaserine is a naturally occurring serinederivative diazo compound and is a known carcinogen. Azaserine is aglutamine analogue that irreversibly inhibits glutamine phosphoribosylamidotransferase, which is involved in the biosynthesis of inosinemonophosphate (IMP). IMP is an important precursor to the purinenucleotides which include adenosine monophosphate (AMP) and guanosinemonophosphate (GMP).

Ascorbic acid (C₆H₈O₆). Ascorbic acid is a sugar acid with antioxidantproperties. L-Ascorbic acid is also known as vitamin C.

Butyric acid (C₄H₈O₂). Butyric acid is a carboxylic acid and a shortchain fatty acid. Butyric acid has been associated with the ability toinhibit the function of histone deacetylase enzymes, thereby favoring anacetylated state of histones in the cell.

Xylose (C₅H₁₀O₅). Xylose (wood sugar) is a five-carbon monosaccharide.Xylose can be metabolized into useful products by a variety oforganisms, e.g., Clostridium difficile.

Sorbitol (C₆H₁₄O₆). Sorbitol, also known as glucitol, is a sugaralcohol. Sorbitol also is an osmotic stress agent (osmotic shock isinduced by 0.5 M sorbitol).

TABLE 2 Additives Compounds (Concentration) Concentration Range SingleConcentration Chromium trioxide 40-60 mg/L 50 mg/L Clindamycin 0.1-10mg/L (e.g., 0.4-0.6 mg/L) 0.5 mg/L Azaserine, 15-50 μM 0.5-1. mM 40-60nM 50 μM 1 mM 50 nM Adenosine, and (e.g., 0.8-1.2 mM) Biotin Ascorbicacid 2.5-10 g/L 2.5 g/L Butyric acid 30-60 nM 60 mM D(+)Xylose 1-15 g/L(e.g., 6-10 g/L) 6 g/L D-Sorbitol 6-20 g/L (e.g., 8-18 g/L) 12 g/LMethods

Growth of C. difficile according to the methods of the inventionproceeds in at least two phases: seed growth and fermentation. The seedgrowth phase, as described further below, may proceed in one or moreseed culture stages (e.g, two stages or three stages).

A seed culture is first grown by inoculating seed medium with a samplefrom a stock culture (e.g., a working cell bank (WCB)). A sample of thisseed culture is used either to inoculate a second seed culture or toinoculate a relatively large fermentation culture. Such seed culturesare typically carried out to allow the quantity of the microorganismfrom a stored culture (e.g., WCB) to be exponentially increased(scaled-up). Seed cultures can also be used to rejuvenate relativelydormant microbes in stored cultures. As is well understood in the art,more than one seed culture (e.g., two or three cultures or stages) canbe used to scale-up the quantity of C. difficile for inoculation intothe fermentation medium.

The number of seed cultures (or stages) used depends on, for example,the size and volume of the fermentation step. For example, the cultureprocess may involve two seed cultures: a first seed culture is grownfrom an inoculation of a WCB (stage one seed culture), a sample of thisseed culture is used to inoculate a second seed culture (stage two seedculture), and a sample from this second culture is used to inoculate afermentation culture (fermentation stage). In a preferred embodiment ofthe present invention, the first and second seed cultures are grown inSYS media.

In stage one, a culture of C. difficile is suspended in seed medium andis incubated at a temperature between 30-40° C., preferably at 37±1° C.,for 18 hours in an anaerobic environment. In stage two, a sample of thestage one seed medium is used to inoculate a stage two seed medium forfurther growth. After inoculation, the stage two medium is incubated ata temperature between 30-40° C., preferably at 37±1° C., forapproximately 10 hours, also in an anaerobic environment. Preferably,growth in seed media at any stage does not result in cell lysis beforeinoculation of fermentation media. Additional growth in a third (fourth,etc.) stage seed culture can also be carried out.

In the fermentation stage, an appropriate concentration of seed culture,which can range from, e.g., 0.1-10%, is used to inoculate thefermentation media. Preferably, concentrations of 1.0% or 5.0% can beused. Most preferably, concentrations of 10% are used.

Fermentation is preferably carried out in an anaerobic chamber atapproximately 35° C. to 45° C. and preferably at a temperature between37° C. to 41° C. (e.g., 37° C.). The pH of the fermentation may becontrolled at a pH between pH 6.35 to 7.45 (e.g., between 6.35 to 6.65,and preferably, at pH 6.5). Alternatively, the pH of the culture mediais uncontrolled and is allowed to decrease naturally during thefermentation process.

C. difficile can be cultivated by fermentation with continuous exposureto a suitable gas or gas mixture (such as, for example, 80% nitrogen/10%CO₂/10% hydrogen, 100% CO₂, or 100% nitrogen). Such gases or gasmixtures may also be sparged (i.e., bubbled) through the medium duringfermentation. As an alternative to sparging (or in addition to it), agas mixture (e.g., 80% nitrogen/10% CO₂/10% hydrogen) or a gas (e.g.,CO₂ or nitrogen) may be applied to the culture media as an overlay todegas the media throughout the fermentation process. The fermentationculture is preferably sparged prior to inoculation with either a mixtureof 80% nitrogen/10% CO₂/10% hydrogen, 100% CO₂ or 100% nitrogen toremove any residual oxygen in the medium. During the fermentationprocess the culture may be sparged periodically. Alternatively, anoverlay of a gas mixture or a gas (e.g., 100% nitrogen) may be appliedto the culture.

Fermentation proceeds for approximately 16 to 24 hours (e.g., 18 to 21hours). Preferably, agitation (e.g., 100 rpm) is applied to the culturemedium during the fermentation process (and/or during stages one and twoof seed cultures). Growth can be monitored by measuring the opticaldensity (O.D.) of the medium.

C. difficile toxins can be isolated and purified from fermentationcultures using purification methods well known in the art such as forexample, Kotloff et al., Infect. Immun 2001; 69:988-995, Coligan et al.,“Current Protocols in Protein Science,” Wiley & Sons; Ozutsumi et al.,Appl. Environ. Microbiol. 49:939-943, 1985; and Kim et al., Infectionand Immunity 55:2984-2992, 1987; which are incorporated herein byreference. The purified toxins can then, for example, be inactivated bychemical treatments known in the art (e.g., formaldehyde treatment).

All references cited within this disclosure are hereby incorporated byreference in their entirety. Certain embodiments are further describedin the following examples. These embodiments are provided as examplesonly and are not intended to limit the scope of the claims in any way.

EXAMPLES

The basal media and additives of the invention were used to cultureClostridium difficile and produce Clostridium difficile Toxins A and B.Tables 3 and 4 (and FIGS. 1A-1G) summarize the amount of toxin producedby Clostridium difficile cells cultured in SYS media with the indicatedadditive at the indicated concentration after the indicated amount oftime. Throughout the examples, Clostridium difficile, ATCC No. 43255,ATCC Lot# 2888434, was cultured.

TABLE 3 Percent increase in Toxins A and B at time points 12 and 24hours following growth in the presence of the listed compounds CompoundsToxin A Toxin B (Concentration) 12 hrs. 24 hrs. 12 hrs. 24 hrs. Chromiumtrioxide (50 mg/L) 18.67 80.45 Clindamycin (0.5 mg/L) 27.52 94.21 50 μMAzaserine + 1 mM 7.69 80.03 96.2 238.34 Adenosine + 50 nM BiotinAscorbic acid (2.5 g/L) 4.65 5.26 24.42 Ascorbic acid (10 g/L) 1.6818.54 49.17 Butyric acid (30 mM) 13.8 10.07 33.6 60.95 Butyric acid (60mM) 88.6 D(+)Xylose (6 g/L) 10.06 8.49 45.83 D-Sorbitol (6 g/L) 49.1986.29 68.03 153.03

TABLE 4A Total measured production of Toxins A and B at time points 12and 24 hours following growth in the presence of the listed compoundsToxin A Toxin B Compounds (ng/mL) (ng/mL) (Concentration) 12 hrs. 24hrs. 12 hrs. 24 hrs. Control (CD-2284) 5974 6809 2436 2645 Chromiumtrioxide 3880 8080 2188 4773 (50 mg/L) Increase (%) 18.67 80.45 Control(CD-2304) 10083 10475 3392 3469 Clindamycin (0.5 mg/L) 611 13358 1896737 Increase (%) 27.52 94.21 Control (CD-2353) 9649 10554 3159 3529 50μM Azaserine, 10391 19000 6198 11940 1 mM Adenosine, 50 nM BiotinIncrease (%) 7.69 80.03 96.20 238.34 Control (CD-2380) 10822 11515 38253992 Ascorbic acid (2.5 g/L) 10789 12050 4026 4967 Increase (%) 4.655.26 24.42 Ascorbic acid (10 g/L) 9957 11708 4534 5955 Increase (%) 1.6818.54 49.17 Butyric acid 12315 12674 5110 6425 (30 mM = 2.75 mL/l)Increase (%) 13.80 10.07 33.60 60.95 Butyric acid 5335 10681 3063 7529(60 mM = 5.5 mL/l) Increase (%) 88.60 Control (CD-2401) 10108 10508 37563832 D(+)Xylose (6 g/L) 10135 11565 4075 5588 Increase (%) 10.06 8.4945.83 D-Sorbitol (6 g/L) 15080 19575 6311 9696 Increase (%) 49.19 86.2968.03 153.03

TABLE 4B Total measured production of Toxins A and B at time points 12and 24 hours following growth in the presence of the listed compoundsToxin A Toxin B Compounds (ng/mL) (ng/mL) (Concentration) 12 hrs. 24hrs. 12 hrs. 24 hrs. 50 μM Azaserine + 10391 19000 6198 11940 1 mMAdenosine + 50 nM Biotin 15 μM Azaserine + 8525 10423 3430 3880 1 mMAdenosine + 50 nM Biotin  5 μM Azaserine + 7471 7816 2665 2694 1 mMAdenosine + 50 nM Biotin

Example 1

This Example includes data on the amount of toxin produced whenClostridium difficile is cultured in SYS basal media in the absence andpresence of various metallic ions.

Materials

The following are the Example 1 test compounds, along with the compoundformula and source.

-   AFC-Ammonium ferric citrate (C₆H₈O₇.nFe.nH₃N), USB Cat. 15751 Lot.    121753-   FC-Ferric citrate (C₆H₅FeO₇), FW 244.95, MB BIomedicals LLC, Cat.    195181, Lot R23927-   FG-Ferrous gluconate Hydrade (C_(12 hrs.22)FeO₁₄)-   FS-Ferric sulfate (FeSO₄.7H₂O), FW 278.02 CA-Calcium chloride    Anhydrous (CaCl₂), FW 110.98, J. T. Baker Cat. 1311-01, Lot. A13602-   CC-Cobalt chloride 6 Hydrate Crystal (CoCl₂.6H₂O), FW 237.93,    Mallinckrodt Chemicals Cat. 4535-02-   CT-Chromium trioxide Crystal (CrO₃), FW 99.99, J. T. Baker Cat.    1638-04, Lot.-   MS-Magnesium sulfate (MgSO₄.7H₂O), FW 246.50-   MC-Manganese chloride (MnCl₂4H₂O), FW 197.90, J. T. Baker Cat.    2540-04, Lot E37335

The following table indicates the natural pH of the indicated compoundin solution at the indicated concentration.

Compound solution Natural Compound solution 2 g/L pH 1 g/L Natural pHAmmonium ferric citrate 5.0 Calcium chloride 4.7 Ferric citrate 3.0Cobalt chloride 4.8 Ferrous gluconate 4.5 Chromium trioxide <2.5 Ferricsulfate 4.7 Magnesium sulfate 5.0 Manganese chloride 5.0Methods

The following methods were used to test the production of Toxin A and Bby Clostridium difficile when cultured in the presence of theabove-listed additives.

I. Medium Preparations:

-   -   1. Prepare 1000 mL SYS medium in 2 L beaker.    -   2. Transfer SYS to media bottles and degas for over 30 minutes        with 10% H₂+10% CO₂+80% N₂.    -   3. Before transferring the medium, fill gas (10% H₂+10% CO₂+80%        N₂) from the fill port of the Flexboy bag into the bag to remove        oxygen, then empty the gas from the bag. Connect the filling        system manifold with the bags.    -   4. For seed medium in 50 mL Flexboy bags, pump 30 mL medium into        the bag from the fill port with a flow speed at 100 mL/minute.    -   5. For fermentation medium in 250 mL Flexboy bags:        -   i) Put the bag on a balance before filling with the medium            and adjust to “0.”        -   ii) Pump the medium into the bag from the fill port with a            flow speed at 100 mL/min until the balance show 50 g, stop            the pumping.    -   6. Move the bag for seed-1 to 37° C. CO₂ incubator to warm        overnight. Keep bag for seed-2 and fermentation at 4° C. until        use.    -   7. Move the bags to 37° C. CO₂ incubator to warm up overnight        before use.    -   8. For different compounds:        -   i) Prepare 40 mL solutions of different compounds with the            concentrations at 2.0 g/L (80 mg compound+40 mL di water            (pH)).        -   ii) Prepare 40 mL solutions of different compounds with the            concentrations at 1.0 g/L (40 mg compound+40 mL di water            (pH)).        -   iii) For all compounds but ferric citrate, filter the            solution using Millipore 50 mL disposable vacuum filtration            system with 0.22 μm Millipore Express Plus membrane. The            ferric citrate was autoclaved.        -   iv) Before transfer of seed-2 to fermentation bags add the            compound solutions as the following concentrations listed in            the following table:

Compounds Sterile di water Total (mg/L) (mL) (mL) 2 g/L solution (mL)Control (without any compound) 0 2.5 2.5 100 mg/L (5 mg/50 mL) 2.5 0 2.51 g/L solution (mL)  50 mg/L (2.5 mg/50 mL) 2.5 0 2.5II. Fermentation Process:

-   -   1. First stage seed culture: 1 mL WCB, containing 50% glycerol,        was transferred into a 50 mL Flexboy bag containing 30 mL SYS        medium and incubated at 37±1° C. for 24 hours.    -   2. Second stage seed culture: 1.5 mL of first stage seed culture        at inoculums of 5% were transferred into the 50 mL Flexboy bag        containing 30 mL SYS medium and incubated at 37±1° C. for 22        hours.    -   3. Fermentation: 2.5 mL of second stage seed culture was        inoculated at 5% for each 250 mL Flexboy bag containing 50 mL of        SYS medium and incubated at 37° C.±1° C. for 24 hours.    -   4. Take samples at 12 hours and 24 hours. Cell growth was        measure at 600 nm. The blank of fermentation media was used as        zero for the spectrophotometer. The cell concentration was        diluted 10×.    -   5. The toxin production is measured by capture ELISA.        III. Capture ELISAs:    -   1. Toxin A standard Lot# CD-2062 (1072506A)    -   2. Goat anti-Toxin A, Lot# CD-2017    -   3. Mouse MAb to C. difficile Toxin A (PCG4)    -   4. Toxin B standard Lot#QC06329    -   5. Goat anti-Toxin B, Lot# C0210091    -   6. Mouse anti Toxin B Lot#030904        Results

-   1. The following table shows the amount of seed growth (OD_(600 nm))    as measured by DU700.

Seed-1 Seed-2 1.73 2.31

-   2. The following table shows the amount of cell growth (OD_(600 nm))    in cultures with the indicated compound.

Test 12 hours 24 hours Control 2.56 2.25 Ammonium ferric citrate 2.582.22* Ferric citrate 2.38 2.48* Ferrous gluconate 2.73 2.51* Ferricsulfate 2.55 2.73* Calcium chloride 2.39 2.20 Cobalt chloride 2.34 1.89*Chromium trioxide 1.48 1.02** Magnesium sulfate 2.37 2.03 Manganesechloride 2.52 1.97 *The broth became dark green because iron reactedwith other compounds **A lot of cells showed 3× to 5× longer than thenormal cells in 24 h broth

-   3. The following table shows the amount of Toxin A produced (ng/mL)    in cultures with the indicated compound (FIGS. 2A and 2B).

Test 12 hours 24 hours Control 5974 6809 Ammonium ferric citrate 57526634 Ferric citrate 5580 6544 Ferrous gluconate 5453 6208 Ferric sulfate5162 5706 Calcium chloride 6294 6563 Cobalt chloride 5252 6647 Chromiumtrioxide 3880 8080 Magnesium sulfate 5060 5527 Manganese chloride 47685449

-   4. The following table shows the amount of Toxin B produced (ng/mL)    in cultures with the indicated compound.

Test 12 hours 24 hours Control 2436 2645 Ammonium ferric citrate 21532503 Ferric citrate 2191 2550 Ferrous gluconate 2190 2082 Ferric sulfate2068 2059 Calcium chloride 2494 2516 Cobalt chloride 2043 2721 Chromiumtrioxide 2188 4773 Magnesium sulfate 1873 1922 Manganese chloride 17841921

-   5. The following table shows the amount of spore formation in 24    hour fermentation in cultures with the indicated compound. Broth was    examined by microscope.

Test 24 hours Control No spore formation found Ammonium ferric citrateNo spore formation found Ferric citrate No spore formation found Ferrousgluconate No spore formation found Ferric sulfate No spore formationfound Calcium chloride No spore formation found Cobalt chloride No sporeformation found Chromium trioxide No spore formation found Magnesiumsulfate No spore formation found Manganese chloride No spore formationfoundConclusions

Chromium trioxide, when added to the SYS medium at 50 mg/L, causedincreases in production of Toxin A (20%) and Toxin B (80%) after 24hours in fermentation broth, but not after 12 hours in fermentationbroth.

Example 2

This example includes data on the amount of toxin produced whenClostridium difficile is cultured in SYS basal media in the absence andpresence of various antibiotics.

Materials

The following are antibiotics, along with the compound formula andsource, which were tested in this example.

-   Cip-Ciprofloxacin (C₁₇H₁₈FN₃O₃) FW 331.35, BioChemika, Lot#WA19781,    soluble with 0.2 mL of 5 N HCl.-   Cli-Clindamycin hydrochloride (C₁₈H₃₃ClN₃O₅S.HCl) FW 461.44, Sigma    C5269, Lot#37k1535, soluble in water.-   Van-Vancomycin hydrochloride (C₆₆H₇₅Cl₂N₉O₂₄.HCl) FW 1485, Sigma    V20029, Lot#037K0686 soluble in water.-   Pen G-Penicillin G Sodium salt (C₁₆H₁₇N₂NaO₄S) FW 356.4, Sigma    P3032, Lot#057K04931, soluble in water.-   Fe-EDTA was also tested (Ethylenediaminetetraacetic acid, Ferric    Sodium Salt, (C₁₀H₁₂FeN₂NaO₈) FW 421.10, Acros Organics 304680050,    Lot#A0245953).

Antibiotics were tested at the following concentrations:

Ciprofloxacin (2 mg/L and 10 mg/L), Clindamycin (0.5 mg/L and 2.5 mg/L),Vancomycin (0.1 mg/L and 0.5 mg/L), and Penicillin G (0.1 mg/L and 0.5mg/L).

Ethylenediaminetetraacetic acid Ferric Sodium Salt was tested at aconcentration of 100 mg/L.

Materials

The following materials were used to test the production of Toxin A andB by Clostridium difficile when cultured in the presence of the aboveantibiotics and compounds.

-   1. Make 100× concentration antibiotic solutions/10× Fe-EDTA    solutions

100× Antibiotics Antibiotics Sterile di Concentration (mg/L) powder (mg)water (mL) (mg/L) Ciprofloxacin (10 mg/L) 40 40 1000 Clindamycin (2.5mg/L) 5 20 250 Vancomycin (0.5 mg/L) 2 40 50 Penicillin G (0.5 mg/L) 240 50 Compounds Compound Sterile di water 10× Concentration (mg/L) (mg)(mL) (mg/L) Fe-EDTA 100 mg/L 40 40 1000

-   2. Make 10× concentration of antibiotic solution:

Take 4 mL of 100× concentration solution+36 mL di water.

Methods

I. Medium Preparations:

-   -   1. Prepare 1000 mL SYS medium in 2 L beaker.    -   2. Transfer SYS to media bottles and degas for over 30 minutes        with 10% H₂+10% CO₂+80% N₂.    -   3. Before transferring the medium, fill gas (10% H₂+10% CO₂+80%        N₂) from the fill port of the Flexboy bag into the bag to remove        oxygen, then empty the gas from the bag. Connect the filling        system manifold with the bags.    -   4. For seed medium in 50 mL Flexboy bags, pump 30 mL medium into        the bag from the fill port with a flow speed at 100 mL/minute.    -   5. For fermentation medium in 250 mL Flexboy bags:        -   i) Put the bag on a balance before filling with the medium            and adjust to “0”.        -   ii) Pump the medium into the bag from the fill port with a            flow speed at 100 mL/min until the balance show 50 g, stop            the pumping.    -   6. Move the bag for seed-1 to 37° C. CO₂ incubator to warm        overnight. Keep bag for seed-2 and fermentation at 4° C. until        use.    -   7. Move the bags to 37° C. CO₂ incubator to warm up overnight        before use.    -   8. For different antibiotics:        -   i) Prepare 40 mL solutions of different antibiotics with the            concentrations at 10× (see above table).        -   ii) Prepare 40 mL solutions of FE-EDTA with the            concentrations at 1000 mg/L (40 mg compound+40 mL di water            (pH)).        -   iii) Filter the solution using the Millipore 50 mL            Disposable Vacuum Filtration System with 0.22 μm Millipore            Express Plus Membrane.        -   iv) Before transfer of seed-2 to fermentation bags, add the            compound solutions at the following concentrations listed in            the following tables.

10× Solution Sterile di Total (mL) water (mL) (mL) Antibiotics (mg/L)Control (without antibiotics) 0 5 5 Ciprofloxacin 2 mg/L 1 4 5 (100μg/50 mL) Ciprofloxacin 10 mg/L 5 0 5 (500 μg/50 mL) Clindamycin 0.5mg/L (25 μg/50 mL) 1 4 5 Clindamycin 2.5 mg/L 5 0 5 (125 μg/50 mL)Vancomycin 0.1 mg/L (5 μg/50 mL) 1 4 5 Vancomycin 0.5 mg/L (25 μg/50 mL)5 0 5 Penicillin G 0.1 mg/L (5 μg/50 mL) 1 4 5 Penicillin G 0.5 mg/L (25μg/50 mL) 5 0 5 Compounds (mg/L) Fe-EDTA 100 mg/L (5 mg/50 mL) 5 0 5II. Fermentation Process:

-   -   1. First stage seed culture: 1 mL WCB, containing 50% glycerol,        was transferred into a 50 mL Flexboy bag containing 30 mL SYS        medium and incubated at 37±1° C. for 24 hours.    -   2. Second stage seed culture: 1.5 mL of first stage seed culture        at inoculums of 5% were transferred into the 50 mL Flexboy bag        containing 30 mL SYS medium and incubated at 37±1° C. for 22        hours.    -   3. Fermentation: 2.5 mL of second stage seed culture was        inoculated at 5% for each 250 mL Flexboy bag containing 50 mL of        SYS medium and incubated at 37° C.±1° C. for 24 hours.    -   4. Take samples at 12 hours and 24 hours. Cell growth was        measure at 600 nm. The blank of fermentation media was used as        zero for the spectrophotometer. The cell concentration was        diluted 10×.    -   5. The toxin production is measured by capture ELISA.        III. Capture ELISAs:    -   1. Toxin A standard Lot# CD-2062 (1072506A)    -   2. Goat anti-Toxin A, Lot# CD-2017    -   3. Mouse MAb to C. difficile Toxin A (PCG4)    -   4. Toxin B standard Lot#QC06329    -   5. Goat anti-Toxin B, Lot# C0210091    -   6. Mouse anti Toxin B Lot#030904        Results

-   1. The following table shows the amount of seed growth (OD_(600 nm))    as measured by DU700.

Seed-1 Seed-2 2.32 2.82

-   2. The following table shows the amount of cell growth (OD_(600 nm))    in cultures with the indicated compound.

Test mg/L 12 hours 24 hours Control — 2.71 2.48 Ciprofloxacin 2 2.602.53 10 0.87 1.38 Clindamycin 0.5 1.24 2.53 2.5 0.11 0.75 Vancomycin 0.13.01 2.53 0.5 3.08 2.52 Penicillin G 0.1 2.69 2.49 0.5 2.64 2.79 Fe-EDTA100 2.91 2.37

-   3. The following table shows the amount of Toxin A produced (ng/mL)    in cultures with the indicated compound (FIGS. 3A and 3B).

Test mg/L 12 hours 24 hours Control — 10083 10475 Ciprofloxacin 2 63096953 10 1685 8614 Clindamycin 0.5 611 13358 2.5 496 445 Vancomycin 0.19142 9557 0.5 8328 9305 Penicillin G 0.1 8435 8871 0.5 8142 8746 Fe-EDTA100 7256 8485

-   4. The following table shows the amount of Toxin B produced (ng/mL)    in cultures with the indicated compound.

Test mg/L 12 hours 24 hours Control — 3392 3469 Ciprofloxacin 2 21352112 10 707 3448 Clindamycin 0.5 189 6737 2.5 146 131 Vancomycin 0.13131 3216 0.5 2770 3127 Penicillin G 0.1 2755 3133 0.5 2816 2664 Fe-EDTA100 2427 2541

-   5. The following table indicates cell morphological characteristics    in cultures with the indicated compound.

Test mg/L 12 hours Control — Normal Ciprofloxacin 2 Normal 10 Many cellswere 2-4× longer than normal cells. Some cells were curved. Clindamycin0.5 Normal 2.5 Most sizes of the cells were 2× smaller than normal.Cells grow very slow Vancomycin 0.1 Some cells were 2× longer thannormal cells 0.5 Some cells were 2× longer than normal cells PenicillinG 0.1 Normal 0.5 Normal Fe-EDTA 100 Normal

-   6. The following table shows the amount of spore formation in 24 h    fermentation in cultures with the indicated compound. Broth was    examined by microscope.

Test mg/L 24 hours Control — No spore formation found Ciprofloxacin 2 Nospore formation found 10 No spore formation found Clindamycin 0.5 Nospore formation found 2.5 No spore formation found Vancomycin 0.1 Nospore formation found 0.5 No spore formation found Penicillin G 0.1 Nospore formation found 0.5 No spore formation found Fe-EDTA 100 No sporeformation foundConclusions

Clindamycin, when added to SYS medium at 0.5 mg/L, caused increases inToxin A (28%) and Toxin B (94%) after 24 hours in fermentation broth,but not after 12 hours in fermentation broth.

Example 3

This Example includes data on the amount of toxin produced whenClostridium difficile is cultured in SYS basal media in the absence andpresence of various vitamins and antibiotics

The following are the Example 3 test compounds, along with the compoundformula and source.

-   Aza-Azaserine (C₅H₇N₃O₄) FW 173.10, (O-diaxoacetyl-L-serine) Fluka    BioChemika, 11430 Lot#1301321, soluble in water.-   Ade-Adenosine (C₁₀H₁₃N₅O₄) FW 267.25, Sigma A4036, Lot#046K06612,    soluble with 5N HCl.-   B₁₂-Vitamin B₁₂ (C₆₃H₈₈CoN₁₄O₁₄P) FW 1,355.37, Sigma,    V6629Lot#124K17072, soluble in water.-   Bio-d-Biotin (C₁₀H₁₆N₂O₃S) FW 244, Supelco 4-7868, Lot#LB5668    soluble with 5N HCl.

The following combinations of compounds were also tested at theindicated concentrations.

#1-50 μM Azaserine (8650 μg/L)+1 mM Adenosine (267 mg/L)+50 nM Biotin(12.2 μg/L)

50 μM Azaserine (432.5 μg/50 mL)+1 mM Adenosine (13.35 mg/50 mL)+50 nMBiotin (610 ng/50 mL)

#2-15 μM Azaserine (2595 μg/L)+1 mM Adenosine (267 mg/L)+50 nM Biotin(12.2 μg/L)

15 μM Azaserine (129.75 μg/50 mL)+1 mM Adenosine (13.35 mg/50 mL)+50 nMBiotin (610 ng/50 mL)

#3-15 μM Azaserine (2595 μg/L), (129.75 μg/50 m/l)

#4-5 μM Azaserine (865 μg/L)+1 mM Adenosine (267 mg/L)+50 nM Biotin(12.2 μg/L)

5 μM Azaserine (43.25 μg/50 mL)+1 mM Adenosine (13.35 mg/50 mL)+50 nMBiotin (610 ng/50 mL)

#5-5 μM Azaserine (865 μg/L)+1 mM Adenosine (267 mg/L)+50 μM Biotin(12.2 ng/L)

5 μM Azaserine (43.25 μg/50 mL)+1 mM Adenosine (13.35 mg/50 mL)+50 μMBiotin (0.61 ng/50 mL)

#6-0.05 nM d-Biotin (12.2 ng/L), (0.61 ng/50 mL)

#7-0.5 nM d-Biotin (122 ng/L), (6.1 ng/50 mL)

#8-5 nM d-Biotin (1.22 μg/L), (61 ng/50 mL)

#9-50 nM Vitamin B12 (67.77 μg/L), (3.39 μg/50 mL)

Materials

1. Make 50× concentration solutions.

50× Chemical Sterile di water Concentration Test component (mg) (mL)(mg/L) Azaserine 4.325 10 432.5 (2.5 mM) (50 μM = 8.65 mg/L) Adenosine133.5 10 13350 (50 mM)  (1 mM = 267 mg/L) 2.5 mM Aza Sterile di water250 μM Azaserine Test component (mL) (mL) (mL) Azaserine 2 18 20 (5 μM =865 μg/L)

2. Make d-Biotin (500 μM) solutions, then dilute to 2.5 μM, 50 nM, 5 nM,and 0.5 nM.

d-Biotin Sterile di 500 μM d-Biotin Test component (mg) water (mL)(mg/L) d-Biotin (50 nM = 12.2 μg/L) 2.44 20 122

3. Make 50× concentration solutions then dilute to 10×.

Sterile di 50× Vitamin B12 water Concentration Test component (mg) (mL)(mg/L) Vitamin B12 33.9 10 3390 (2.5 μM) (50 nM = 67.8 mg/L) 50× 10×concentration Sterile di water Concentration Test component (mL) (mL)(mg/L) Vitamin B12 2 8 678 (500 nM) (50 nM = 67.8 mg/L)MethodsI. Medium Preparations:

-   -   1. Prepare 1000 mL SYS medium in 2 L beaker.    -   2. Transfer SYS to media bottles and degas for over 30 minutes        with 10% H₂+10% CO₂+80% N₂.    -   3. Before transferring the medium, fill gas (10% H₂+10% CO₂+80%        N₂) from the fill port of the Flexboy bag into the bag to remove        oxygen, then empty the gas from the bag. Connect the filling        system manifold with the bags.    -   4. For seed medium in 50 mL Flexboy bags, pump 30 mL medium into        the bag from the fill port with a flow speed at 100 mL/minute.    -   5. For fermentation medium in 250 mL Flexboy bags:        -   i) Put the bag on a balance before filling with the medium            and adjust to “0.”        -   ii) Pump the medium into the bag from the fill port with a            flow speed at 100 mL/minute until the balance show 50 g,            stop the pumping.    -   6. Move the bag for seed-1 to 37° C. CO₂ incubator to warm        overnight. Keep bag for seed-2 and fermentation at 4° C. until        use.    -   7. Move the bags to 37° C. CO₂ incubator to warm up overnight        before use.    -   8. For different compounds:        -   i) Prepare the solutions (see above table).        -   ii) Filter the solution using the Millipore 50 mL Disposable            Vacuum Filtration System with 0.22 μm Millipore Express Plus            Membrane.        -   iii) Before transfer of seed-2 to fermentation bags, add the            compound solutions as the following concentrations listed in            the following tables.

2.5 mM 50 mM 2.5 μM di Chemical add Aza Ade Bio water to 50 mL SYSmedium (mL) (mL) (mL) (mL) Total (mL) Control 0 0 0 5 5 #1-50 μMAzaserine + 1 1 1 2 5 1 mM Adenosine + 50 nM Biotin 250 μM 50 mM 2.5 μMdi Aza Ade Bio water (mL) (mL) (mL) (mL) Total (mL) #2-15 μM Azaserine +3 1 1 0 5 1 mM Adenosine + 50 nM Biotin #3-15 μM Azaserine 3 0 0 2 5#4-5 μM Azaserine + 1 1 1 2 5 1 mM Adenosine + 50 nM Biotin 50 μM 50 mM5 nM di Aza Ade Bio water (mL) (mL) (mL) (mL) Total (mL) #5-5 μMAzaserine + 1 1 0.5 2.5 5 1 mM Adenosine + 50 pM Biotin) 0.5 nM Biotin 5nM Biotin 50 nM Total (mL) (mL) Biotin (mL) (mL) #6-0.05 nM Biotin 5 0 05 #7-0.5 nM Biotin 0 5 0 5 #8-5 nM Biotin 0 0 5 5 500 nM Vitamin B12Total (mL) (mL) #9-50 nM Vitamin B12 5 5 (67.77 μg/L)II. Fermentation Process:

-   -   1. First stage seed culture: 1 mL WCB, containing 50% glycerol,        was transferred into a 50 mL Flexboy bag containing 30 mL SYS        medium and incubated at 37±1° C. for 24 hours.    -   2. Second stage seed culture: 1.5 mL of first stage seed culture        at inoculums of 5% were transferred into the 50 mL Flexboy bag        containing 30 mL SYS medium and incubated at 37±1° C. for 22        hours.    -   3. Fermentation: 2.5 mL of second stage seed culture was        inoculated at 5% for each 250 mL Flexboy bag containing 50 mL of        SYS medium and incubated at 37° C.±1° C. for 24 hrs.    -   4. Take samples at 12 hours and 24 hours. Cell growth was        measure at 600 nm. The blank of fermentation media was used as        zero for the spectrophotometer. The cell concentration was        diluted 10×.    -   5. The toxin production is measured by capture ELISA.        III. Capture ELISAs:    -   1. Toxin A standard Lot# CD-2062 (1072506A)    -   2. Goat anti-Toxin A, Lot# CD-2017    -   3. Mouse MAb to C. difficile Toxin A (PCG4)    -   4. Toxin B standard Lot#QC06329    -   5. Goat anti-Toxin B, Lot# C0210091    -   6. Mouse anti Toxin B Lot#030904        Results

-   1. The following table shows the amount of seed growth (OD_(600 nm))    as measured by DU700.

Seed-1 Seed-2 2.53 2.49

-   2. The following table shows the amount of cell growth (OD_(600 nm))    in cultures with the indicated compound.

Test 12 hours 24 hours Control 2.71 2.39 #1-50 μM Azaserine + 1 mMAdenosine + 1.19 0.42 50 nM Biotin #2-15 μM Azaserine + 1 mM Adenosine +2.76 2.02 50 nM Biotin #3-15 μM Azaserine 2.33 2.55 #4-5 μM Azaserine +1 mM Adenosine + 2.84 2.48 50 nM Biotin #5-5 μM Azaserine + 1 mMAdenosine + 2.49 2.47 50 pM Biotin #6 0.05 nM Biotin 3.03 2.74 #7 0.5 nMBiotin 2.59 2.51 #8 5 nM Biotin 2.86 2.54 #9 50 nM Vitamin B12 2.82 2.88

-   3. The following table shows the amount of Toxin A produced (ng/mL)    in cultures with the indicated compound.

Test 12 hours 24 hours Control 9649 10554 #1-50 μM Azaserine + 1 mMAdenosine + 10391 19000 50 nM Biotin #2-15 μM Azaserine + 1 mMAdenosine + 8525 10423 50 nM Biotin #3-15 μM Azaserine 9333 10838 #4-5μM Azaserine + 1 mM Adenosine + 7471 7816 50 nM Biotin #5-5 μMAzaserine + 1 mM Adenosine + 9933 10811 50 pM Biotin #6 0.05 nM Biotin8708 9481 #7 0.5 nM Biotin 8601 9124 #8 5 nM Biotin 8573 8877 #9 50 nMVitamin B12 5858 6286

-   4. The following table shows the amount of Toxin B produced (ng/mL)    in cultures with the indicated compound.

Test 12 hours 24 hours Control 3159 3529 #1-50 μM Azaserine + 1 mMAdenosine + 6198 11940 50 nM Biotin #2-15 μM Azaserine + 1 mMAdenosine + 3430 3880 50 nM Biotin #3-15 μM Azaserine 3589 4112 #4-5 μMAzaserine + 1 mM Adenosine + 2665 2694 50 nM Biotin #5-5 μM Azaserine +1 mM Adenosine + 3345 3813 50 pM Biotin #6 0.05 nM Biotin 2616 3167 #70.5 nM Biotin 2717 3084 #8 5 nM Biotin 2756 2936 #9 50 nM Vitamin B121819 2030Conclusions

50 μM Azaserine, 1 mM Adenosine, and 50 nM Biotin together, when addedto the SYS medium, caused increases in production of Toxin A (80%) andToxin B (238%) after 24 hours in fermentation broth.

Example 4

This Example includes data on the amount of toxin produced whenClostridium difficile is cultured in SYS basal media in the absence andpresence of various amino acids and organic compounds.

Materials

The following are the Example 4 test compounds, along with the compoundformula and source.

-   -   Arg-L-Arginine Monohydrochloride (C₆H₄N₄O₂.HCl), FW. 210.67        Sigma A5131-500G, Lot# 016K0001, soluble in water.    -   Cys-L-Cysteine (C₃H₇NO₂S), FW 121.16, Sigma C-7352,        Lot#082K0377, soluble in water.    -   Tyr-L-Tyrosine (C₉H₁₁NO₃), FW 181.19, Sigma T8566, Lot#107K0157,        soluble in water with HCl.    -   Asc-Ascorbic acid (C₆H₈O₆) FW 176.12, Sigma A5960 Lot#043K0131,        soluble in water.    -   But-Butyric acid (C₄H₈O₂) FW 88.11, Aldrich B103500 Lot#03511DA,        soluble in water.

These compounds were tested using the following concentrations (10×):

L-Arginine Monohydrochloride (50 mM).

L-Tyrosine (50 mg/L).

L-Cysteine (0.33 mM, 10 mM, and 33 mM).

Ascorbic acid (2.5 g/L and 10 g/L).

Butyric acid (30 mM and 60 mM).

-   1. Make 10× Arginine solutions.

Sterile 10× Arginine di water Concentration Test component (g) (mL)(g/L) Arginine (50 mM = 10.5 g/L) 4.2 40 105

-   2. Make 10× Cysteine solutions at 33 mM then dilute to 1 mM and 0.33    mM.

Sterile 10× di water Concentration Test component Cysteine (g) (mL)(g/L) Cysteine (33 mM = 4 g/L) 1.6 40 40 10× Sterile 10× 33 mM Cys. diwater Concentration Test component (mL) (mL) (g/L) Cysteine (3.3 mM =400 mg/L) 4 36 4 10× Sterile 10× 3.3 mM Cys. di water Concentration Testcomponent (mL) (mL) (g/L) Cysteine (0.33 mM = 40 mg/L) 4 36 0.4

-   3. Make 10× Tyrosine solutions at 50 mg/L.

Tyrosine Sterile di water 10× Concentration Test component (g) (mL)(g/L) Tyrosine (50 mg/L) 0.02 40 1

-   4. Make 10× Ascorbic acid solutions at 2.5 g/L and 10 g/L.

Sterile Ascorbic acid di water 10× Concentration Test component (g) (mL)(g/L) Ascorbic acid (2.5 g/L) 1 40 25 Ascorbic acid (10 g/L) 4 40 100

-   5. Make 10× Butyric acid solutions at 30 mM.

Butyric acid Sterile di water 10× Concentration Test component (mL) (mL)(mL/l) Butyric acid 1.1 38.9 27.5 (30 mM = 2.75 mL/L) Butyric acid 2.237.8 55 (60 mM = 5.5 mL/L)MethodsI. Medium Preparations:

-   -   1. Prepare 1000 mL SYS medium in 2 L beaker.    -   2. Transfer SYS to media bottles and degas for over 30 minutes        with 10% H₂+10% CO₂+80% N₂.    -   3. Before transferring the medium, fill gas (10% H₂+10% CO₂+80%        N₂) from the fill port of the Flexboy bag into the bag to remove        oxygen, then empty the gas from the bag. Connect the filling        system manifold with the bags.    -   4. For seed medium in 50 mL Flexboy bags, pump 30 mL medium into        the bag from the fill port with a flow speed at 100 mL/minute.    -   5. For fermentation medium in 250 mL Flexboy bags:        -   i) Put the bag on a balance before filling with the medium            and adjust to “0”.        -   ii) Pump the medium into the bag from the fill port with a            flow speed at 100 mL/min until the balance show 50 g, stop            the pumping.    -   6. Move the bag for seed-1 to 37° C. CO₂ incubator to warm        overnight. Keep bag for seed-2 and fermentation at 4° C. until        use.    -   7. Move the bags to 37° C. CO₂ incubator to warm up overnight        before use.    -   8. For different antibiotics:        -   i) Prepare 40 mL solutions of different antibiotics with the            concentrations at 10× (see above table).        -   ii) Prepare 40 mL solutions of FE-EDTA with the            concentrations at 1000 mg/L (40 mg compound+40 mL di water            (pH)).        -   iii) Filter the solution using the Millipore 50 mL            Disposable Vacuum Filtration System with 0.22 μm Millipore            Express Plus Membrane.        -   iv) Before transfer of seed-2 to fermentation bags add the            compound solutions at the concentrations listed in the            following tables.

10× Sterile di Medium Solution water Total # Test compound (mL) (mL)(mL) 0 Control (without antibiotics) 0 5 5 1 Arginine (50 mM = 10.5 g/L)5 0 5 2 Cysteine (0.33 mM = 40 mg/L) 5 0 5 3 Cysteine (3.3 mM = 400mg/L) 5 0 5 4 Cysteine (33 mM = 4 g/L) 5 0 5 5 Tyrosine (50 mg/L) 5 0 56 Ascorbic acid (2.5 g/L) 5 0 5 7 Ascorbic acid (10 g/L) 5 0 5 8 Butyricacid (30 mM = 2.75 mL/l) 5 0 5 9 Butyric acid (60 mM = 5.5 mL/l) 5 0 5II. Fermentation Process:

-   -   1. First stage seed culture: 1 mL WCB, containing 50% glycerol,        was transferred into a 50 mL Flexboy bag containing 30 mL SYS        medium and incubated at 37±1° C. for 24 hours.    -   2. Second stage seed culture: 1.5 mL of first stage seed culture        at inoculums of 5% were transferred into the 50 mL Flexboy bag        containing 30 mL SYS medium and incubated at 37±1° C. for 22        hours.    -   3. Fermentation: 2.5 mL of second stage seed culture was        inoculated at 5% for each 250 mL Flexboy bag containing 50 mL of        SYS medium and incubated at 37° C.±1° C. for 24 hrs.    -   4. Take samples at 12 hours and 24 hours. Cell growth was        measure at 600 nm. The blank of fermentation media was used as        zero for the spectrophotometer. The cell concentration was        diluted 10×.    -   5. The toxin production is measured by capture ELISA.        III. Capture ELISAs:    -   1. Toxin A standard Lot# CD-2062 (1072506A)    -   2. Goat anti-Toxin A, Lot# CD-2017    -   3. Mouse MAb to C. difficile Toxin A (PCG4)    -   4. Toxin B standard Lot#QC06329    -   5. Goat anti-Toxin B, Lot# C0210091    -   6. Mouse anti Toxin B Lot#030904        Results

-   1. The following table shows the amount of seed growth (OD_(600 nm))    as measured by DU700.

Seed-1 Seed-2 2.65 2.53

-   2. The following table shows the amount of cell growth (OD_(600 nm))    in cultures with the indicated compound.

Test 12 hours 24 hours Control 2.61 2.61 #1 Arginine (50 mM = 10.5 g/L)2.89 2.55 #2 Cysteine (0.33 mM = 40 mg/L) 2.43 2.36 #3 Cysteine (3.3 mM= 400 mg/L) 2.38 2.59 #4 Cysteine (33 mM = 4 g/L) 1.67 1.74 #5 Tyrosine(50 mg/L) 2.41 2.34 #6 Ascorbic acid (2.5 g/L) 2.49 2.19 #7 Ascorbicacid (10 g/L) 2.16 1.98 #8 Butyric acid (30 mM = 2.75 mL/l) 2.37 2.00 #9Butyric acid (60 mM = 5.5 mL/l) 1.38 1.98

-   3. The following table shows the amount of Toxin A produced (ng/mL)    in cultures with the indicated compound (FIGS. 4A and 4B).

Test 12 hours 24 hours Control 10822 11515 #1 Arginine (50 mM = 10.5g/L) 6007 6616 #2 Cysteine (0.33 mM = 40 mg/L) 9691 10365 #3 Cysteine(3.3 mM = 400 mg/L) 9828 10741 #4 Cysteine (33 mM = 4 g/L) 897 853 #5Tyrosine (50 mg/L) 11394 11624 #6 Ascorbic acid (2.5 g/L) 10789 12050 #7Ascorbic acid (10 g/L) 9957 11708 #8 Butyric acid (30 mM = 2.75 mL/l)12315 12674 #9 Butyric acid (60 mM = 5.5 mL/l) 5335 10681

-   4. The following table shows the amount of Toxin B produced (ng/mL)    in cultures with the indicated compound.

Test 12 hours 24 hours Control 3825 3992 #1 Arginine (50 mM = 10.5 g/L)2300 2626 #2 Cysteine (0.33 mM = 40 mg/L) 3446 3581 #3 Cysteine (3.3 mM= 400 mg/L) 3017 3185 #4 Cysteine (33 mM = 4 g/L) 339 322 #5 Tyrosine(50 mg/L) 3752 4462 #6 Ascorbic acid (2.5 g/L) 4026 4967 #7 Ascorbicacid (10 g/L) 4534 5955 #8 Butyric acid (30 mM = 2.75 mL/l) 5110 6425 #9Butyric acid (60 mM = 5.5 mL/l) 3063 7529

-   5. The following table indicates cell morphological characteristics    in cultures with the indicated compound.

Test 12 hours/24 hours Control Normal #1 Arginine (50 mM = 10.5 g/L)Normal #2 Cysteine (0.33 mM = 40 mg/L) Normal #3 Cysteine (3.3 mM = 400mg/L) Normal #4 Cysteine (33 mM = 4 g/L) Cells show gray color #5Tyrosine (50 mg/L) Normal #6 Ascorbic acid (2.5 g/L) Normal #7 Ascorbicacid (10 g/L) Normal #8 Butyric acid (30 mM = 2.75 mL/l) Normal #9Butyric acid (60 mM = 5.5 mL/l) Normal

-   6. The following table shows the amount of spore formation in 24    hour fermentation in cultures with the indicated compound. Broth was    examined by microscope.

Test Spore formation Control No spore found #1 Arginine (50 mM = 10.5g/L) Very a few spores found #2 Cysteine (0.33 mM = 40 mg/L) Very a fewspores found #3 Cysteine (3.3 mM = 400 mg/L) No spore found #4 Cysteine(33 mM = 4 g/L) No spore found #5 Tyrosine (50 mg/L) No spore found #6Ascorbic acid (2.5 g/L) No spore found #7 Ascorbic acid (10 g/L) Nospore found #8 Butyric acid (30 mM = 2.75 mL/l) No spore found #9Butyric acid (60 mM = 5.5 mL/l) No spore foundConclusions

Ascorbic acid, when added to SYS medium at 10 g/L, caused increases inToxin B of 19% after 12 hours and 49% after 24 hours of incubation infermentation broth.

Butyric Acid, when added to SYS medium at 30 mM, caused increases inToxin A of 14% and Toxin B of 34% after 12 hours of incubation infermentation broth. It caused increases in Toxin A of 16% and Toxin B of61% after 24 hours of incubation in fermentation broth. Butyric Acid,when added to SYS medium at 60 mM, caused increases in Toxin B of 89%after 24 hours of incubation in fermentation broth.

Example 5

This Example includes data on the amount of toxin produced whenClostridium difficile is cultured in SYS basal media in the absence andpresence of various concentrations of carbohydrates.

The following table summarizes the data regarding toxin increases (%)following the addition of increasing concentrations of D-sorbitol.

D-Sorbitol Toxin A Toxin B Concentration 12 hours 24 hours 12 hours 24hours  6 g/L 25.55 53.98 45.99 112.30  8 g/L 25.09 74.99 43.39 148.98 10g/L 45.47 140.89 51.76 216.31 12 g/L 46.80 150.04 59.21 295.51 14 g/L23.50 127.69 27.77 254.23 16 g/L 14.91 117.43 21.49 244.44 18 g/L 34.90136.02 30.00 236.74 20 g/L 8.57 118.43 9.90 212.09Materials

The following are the Example 5 test compounds, along with the compoundformula and source.

D(−)Fructose: (contained <0.05% glucose) C₆H₁₂O₆, FW 180.2, Sigma F0127Lot#60K0013

D(+)Galactose: C₆H₁₂O₆, FW 180.2, Sigma G0625, Lot#102K0169 soluble inwater (1 g/1.7 mL)

D(+)Mannose: C₆H₁₂O₆, FW 180.16, Sigma M6020 Lot# soluble in water (50mg/mL)

D(+)Maltose Monohydrate: (contained <0.3% glucose),C_(12 hrs.22)O₁₁.H₂O, FW 360.3, Sigma M9171 Lot#80K10101 soluble inwater

Sucrose: C_(12 hrs.22)O₁₁, FW 342.3, Sigma, 53929, Lot#127K0093 solublein water

-   -   α-Lactose: C_(12 hrs.22)O₁₁.H₂O, FW 360.3, Sigma L2643, Lot#        soluble in water (0.2 g/mL)

D(+)Xylose: C₅H₁₀O₅, FW 150.132, Sigma X3877 Lot# soluble in water (1g/0.8 mL)

D-Sorbitol: C₆H₁₄O₆, FW 182.2, Sigma 53889, Lot#042K01355 soluble inwater.

myo-Inositol: C₆H₁₂O₆, FW 180.16, Sigma 17508, Lot# soluble in water (50mg/mL)

10× solutions of the above carbohydrates were produced.

Methods

I. Medium Preparations:

-   -   1. Prepare 1000 mL SYS medium in 2 L beaker.    -   2. Transfer SYS to media bottles and degas for over 30 minutes        with 10% H₂+10% CO₂+80% N₂.    -   3. Before transferring the medium, fill gas (10% H₂+10% CO₂+80%        N₂) from the fill port of the Flexboy bag into the bag to remove        oxygen, then empty the gas from the bag. Connect the filling        system manifold with the bags.    -   4. For seed medium in 50 mL Flexboy bags, pump 30 mL medium into        the bag from the fill port with a flow speed at 100 mL/minute.    -   5. For fermentation medium in 250 mL Flexboy bags:        -   i) Put the bag on a balance before filling with the medium            and adjust to “0.”        -   ii) Pump the medium into the bag from the fill port with a            flow speed at 100 mL/min until the balance show 50 g, stop            the pumping.    -   6. Move the bag for seed-1 to 37° C. CO₂ incubator to warm        overnight. Keep bag for seed-2 and fermentation at 4° C. until        use.    -   7. Move the bags to 37° C. CO₂ incubator to warm up overnight        before use.    -   8. For different compounds test:        -   i) Prepare the solutions with different chemicals (see above            table)        -   ii) Filter the solution using Millipore 50 mL Disposable            Vacuum Filtration System with 0.22 μm Millipore Express Plus            Membrane.        -   iii) Before transfer of seed-2 to fermentation bags, add the            compound solutions as follows:

10× Sterile Solution di water Total Medium # Test compound (mL) (mL)(mL) 0 Control (without carbohydrate 0 5 5 additive) 1 D(−)Fructose (6g/L) 5 0 5 2 D(+)Galactose (6 g/L) 5 0 5 3 D(+)Mannose (6 g/L) 5 0 5 4D(+)Maltose Monohydrate (6 g/L) 5 0 5 5 Sucrose (6 g/L) 5 0 5 6α-Lactose (6 g/L) 5 0 5 7 D(+)Xylose (6 g/L) 5 0 5 8 D-Sorbitol (6 g/L)5 0 5 9 myo-Inositol (6 g/L) 5 0 5II. Fermentation Process:

-   -   1. First stage seed culture: 1 mL WCB, containing 50% glycerol,        was transferred into a 50 mL Flexboy bag containing 30 mL SYS        medium and incubated at 37±1° C. for 24 hours.    -   2. Second stage seed culture: 1.5 mL of first stage seed culture        at inoculums of 5% were transferred into the 50 mL Flexboy bag        containing 30 mL SYS medium and incubated at 37±1° C. for 22        hours.    -   3. Fermentation: 2.5 mL of second stage seed culture was        inoculated at 5% for each 250 mL Flexboy bag containing 50 mL of        SYS medium and incubated at 37° C.±1° C. for 24 hours    -   4. Take samples at 12 hours and 24 hours. Cell growth was        measure at 600 nm. The blank of fermentation media was used as        zero for the spectrophotometer. The cell concentration was        diluted 10×.    -   5. The toxin production is measured by capture ELISA.        III. Capture ELISAs:    -   1. Toxin A standard Lot# CD-2062 (1072506A)    -   2. Goat anti-Toxin A, Lot# CD-2017    -   3. Mouse MAb to C. difficile Toxin A (PCG4)    -   4. Toxin B standard Lot#QC06329    -   5. Goat anti-Toxin B, Lot# C0210091    -   6. Mouse anti Toxin B Lot#030904        Results

-   1. The following table shows the amount of seed growth (OD_(600 nm))    as measured by DU700.

Seed-1 Seed-2 2.57 2.561

-   2. The following table shows the amount of cell growth (OD_(600 nm))    in cultures with the indicated compound.

Test 12 hours 24 hours Control (without carbohydrate additive) 2.78 2.57#1 D(−)Fructose (6 g/L) 3.42 2.21 #2 D(+)Galactose (6 g/L) 2.92 2.51 #3D(+)Mannose (6 g/L) 5.14 2.84 #4 D(+)Maltose Monohydrate (6 g/L) 2.912.80 #5 Sucrose (6 g/L) 2.74 2.62 #6 α-Lactose (6 g/L) 2.57 2.36 #7D(+)Xylose (6 g/L) 3.34 3.53 #8 D-Sorbitol (6 g/L) 4.00 3.35 #9myo-Inositol (6 g/L) 2.61 2.53

-   3. The following table shows the amount of Toxin A produced (ng/mL)    in cultures with the indicated compound (FIGS. 5A and 5B).

Test 12 hours 24 hours Control 10108 10508 #1 D(−)Fructose (6 g/L) 10312146 #2 D(+)Galactose (6 g/L) 8596 9154 #3 D(+)Mannose (6 g/L) 3568 5288#4 D(+)Maltose Monohydrate (6 g/L) 8741 8764 #5 Sucrose (6 g/L) 1104210881 #6 α-Lactose (6 g/L) 9258 10167 #7 D(+)Xylose (6 g/L) 10135 11565#8 D-Sorbitol (6 g/L) 15080 19575 #9 myo-Inositol (6 g/L) 8189 8743

-   4. The following table shows the amount of Toxin B produced (ng/mL)    in cultures with the indicated compound.

Test 12 hours 24 hours Control 3756 3832 #1 D(−)Fructose (6 g/L) 319 755#2 D(+)Galactose (6 g/L) 3071 3379 #3 D(+)Mannose (6 g/L) 1518 2123 #4D(+)Maltose Monohydrate (6 g/L) 3079 3022 #5 Sucrose (6 g/L) 3902 4235#6 α-Lactose (6 g/L) 3126 3655 #7 D(+)Xylose (6 g/L) 4075 5588 #8D-Sorbitol (6 g/L) 6311 9696 #9 myo-Inositol (6 g/L) 2743 3196

-   5. The following table indicates cell morphological characteristics    in cultures with the indicated compound.

Test 12 hours/24 hours Control Normal #1 D(−)Fructose (6 g/L) Cell lyseat 24 hours #2 D(+)Galactose (6 g/L) Normal #3 D(+)Mannose (6 g/L) Celllyse at 24 hours #4 D(+)Maltose Monohydrate (6 g/L) Normal #5 Sucrose (6g/L) Normal #6 α -Lactose (6 g/L) Normal #7 D(+)Xylose (6 g/L) Normal #8D-Sorbitol (6 g/L) Cell lyse at 24 hours #9 myo-Inositol (6 g/L) Normal

-   6. The following table shows the amount of spore formation in 24 h    fermentation in cultures with the indicated compound. Broth was    examined by microscope.

Test Spore formation Control No spore found #1 D(−)Fructose (6 g/L) Nospore found #2 D(+)Galactose (6 g/L) No spore found #3 D(+)Mannose (6g/L) No spore found #4 D(+)Maltose Monohydrate (6 g/L) No spore found #5Sucrose (6 g/L) No spore found #6 α -Lactose (6 g/L) No spore found #7D(+)Xylose (6 g/L) No spore found #8 D-Sorbitol (6 g/L) No spore found#9 myo-Inositol (6 g/L) No spore foundConclusions

D(+)Xylose, when added to SYS medium at 6 g/L, slightly increased cellgrowth and Toxin A production. D(+)Xylose increased Toxin B production9% after 12 hours of incubation in fermentation broth and 46% after 24hours of incubation in fermentation broth.

D-Sorbitol, when added to SYS medium at 6 g/L, markedly increased cellgrowth and toxins production. Cell growth was increased 44% after 12hours of incubation in fermentation broth. Toxin A production wasincreased 49% after 12 hours of incubation in fermentation broth and 86%after 24 hours of incubation in fermentation broth. Toxin B productionwas increased 68% after 12 hours of incubation in fermentation broth and153% after 24 hours of incubation in fermentation broth.

Example 6

This example includes data on the amount of toxin produced whenClostridium difficile is cultured in SYS basal media supplemented withD-Sorbitol (12 g/L) under pH controlled conditions.

Materials

The following materials and equipment were used in this example.

A Biostat B Plus Fermentation System (Sartorius) was used for the firststage seed culture and second stage seed culture, using 2×2 L fermenters(Sartorius), one for each seed culture seed. A Biostat Q PlusFermentation System (Sartorius) was used for the third stage seedculture using 4×1 L fermenters (Sartorius). A peristalitic pump(Masterflex) was used to transfer media for both systems. Mediacomposition for the seed stages were as follows:

SYS media Formulation Component Manufacturer/Lot # (g/L) KH₂PO₄ J TBaker/E29H22 0.9 Na₂PO₄ J T Baker/A12145 5 NaHCO₃ J T Baker/A13668 5 SoyPeptone A3 SC Organotechnie/19685 30 Yeast Extract BD Bacto/7109497 20Media composition for the production stage was as follows:

SYS media + Sorbitol Formulation Component Manufacturer/Lot # (g/L)KH₂PO₄ J T Baker/E29H22 0.9 Na₂PO₄ J T Baker/A12145 5 NaHCO₃ J TBaker/A13668 5 Soy Peptone A3 SC Organotechnie/19685 30 Yeast Extract BDBacto/7109497 20 D-Sorbitol(70%)* Spectrum/WJ1030 17.1 ml *17.1 ml ofD-Sorbitol (70%) represents 12 g/LThe SYS media and SYS media+Sorbitol were each prepared using reverseosmosis deionized water (RODI-water).Additional materials included:

Qty. Manufacturer/Lot # Part # Added Working Cell In-house n/a 0.5 (2ml) Bank vial, WCB-A, 4.5 ml Anaerobic Gas RM-0024 20 mix(80% N2/10%CO2/10% H2) 5N Sodium J T Baker/E17507 5671-06 1 L Hydroxide 1NHydrochloric J T Baker/B08510 5618-02 200 ml AcidMethodsThe following methods were used to test the production of Toxin A and Bwhen cultured under pH controlled conditions.I. Seed Bioreactor 1

-   1. A 2 L vessel was prepared with a ring sparger and a pitched blade    impeller on the bottom of the shaft set at a 45° angle.-   2. A pH probe was calibrated according to Sartorius procedures and    installed in the bioreactor.-   3. The bioreactor was then autoclaved on a dry cycle for 30 min with    10 min pre and post-vacuum cycles.-   4. After sterilization, the sterile vessel was connected to the    Biostat B Plus System.-   5. SYS medium was prepared as described above-   6. 1600 mL of medium were aseptically transferred to the sterile    bioreactor.-   7. Vessel temperature and agitation were set to 37° C. and 100 rpm,    respectively.-   8. Prior to inoculation, the bioreactor was de-gassed by sparging    with anaerobic gas mix at 300 mL/min for 15 minutes.-   9. 4 mL of WCB-A was aseptically transferred to the bioreactor to    initiate the culture.-   10. During the culture, the bioreactor was sparged with anaerobic    gas mix at 100 mL/min and incubated for 18 h.-   11. At end of 18 hr, a 5 ml sample was taken for OD measurement.    II. Seed Bioreactor 2-   1. A 2 L vessel was prepared with a ring sparger and a pitched blade    impeller on the bottom of the shaft set at a 45° angle.-   2. A pH probe was calibrated according to Sartorius procedures and    installed in the bioreactor.-   3. The bioreactor was then autoclaved on a dry cycle for 30 min with    10 min pre and post-vacuum cycles.-   4. After sterilization, the sterile vessel was connected to the    Biostat B Plus System.-   5. SYS medium was prepared as described above-   6. 1800 mL of medium were aseptically transferred to the sterile    bioreactor.-   7. Vessel temperature and agitation were set to 37° C. and 100 rpm,    respectively.-   8. Prior to inoculation, medium in vessel was sparged with anaerobic    gas mix at 300 mL/min for 15 minutes.-   9. 100 mL of the 1st stage culture was aseptically transferred the    2nd stage.-   10. During the culture, the bioreactor was sparged with anaerobic    gas mix at 100 mL/min and incubated for 10 h.-   11. At end of 10 hr, a 5 ml sample was taken for OD measurement.    III. Production Bioreactor-   1. 4×1 L vessels were prepared each with a ring sparger and a    pitched blade impeller on the bottom of the shaft set at ˜45° angle.-   2. A pH probe for each bioreactor was calibrated according to    Sartorius procedures and installed in each bioreactor.-   3. All bioreactors were then autoclaved on a dry cycle for 30 min    with 10 min pre and post-vacuum cycles.-   4. After sterilization, the bioreactors were connected to the    Biostat Q Plus System.-   5. SYS medium+Sorbitol was prepared as described above-   6. 900 mL of medium was aseptically transferred to each bioreactor.-   7. Acid and base bottles were autoclaved, aseptically filled with    sterile filtered 1N HCl and 5N NaOH, respectively, and attached to    the bioreactors.-   8. Agitiaton was set to 100 rpm for all bioreactors.-   9. The desired temperature and pH control set points were    implemented (see Table 5).-   10. Prior to inoculation, bioreactors were de-gassed by sparging    with appropriate gas (see Table 5) for 30 minutes at 300 mL/min.-   11. Each vessel was inoculated with ˜100 mL of culture from Seed    Bioreactor 2.-   12. 3^(rd) stage cultures were incubated at 37° C. with no    additional sparging for 18 hours.-   13. Samples (˜5 mL) were taken at appropriate times for OD and toxin    measurements, typically between 14.5 to 18 hrs post-inoculation.-   14. For ELISA, 2×1 mL of sample were spun in 1.8 mL microcentrifuge    tubes at 10,000 g for 1 min, then decanted and 0.2 μm filtered. The    samples were stored at 2-8° C. until tested.

TABLE 5 Vessel Volume Inoc Temp Stirrer pH NaHCO₃ Degas Sparge CultureNo. (ml) (ml) ° C. (rpm) Control* (g/L) Gas Gas Stage B1 800  2 37 100None 5 Gas mix Gas mix 1^(st) stage B2 900 100 37 100 None 5 Gas mix Gasmix 2^(nd) stage QA1 900 100 37 100 6.5 5 Gas mix None 3^(rd) stage QA2900 100 37 100 7.2 5 Gas mix None 3^(rd) stage QA3 900 100 37 100 8.0 5Gas mix None 3^(rd) stage QB3 900 100 37 100 None 5 Gas mix None 3^(rd)stage *pH control using 1N HCl, 5N NaOHResults

The following table shows total cell growth (OD 600 nm) and the amountof Toxin A produced (ng/ml) and Toxin B produced (ng/ml) in the culturessubject to the indicated pH control (FIGS. 6A, 6B).

Toxin A Toxin B Sample OD@600 nm (ng/ml) (ng/ml) 1 stage @18 hrs 1.96N/A N/A 2^(nd) stage @10 hrs 2.71 N/A N/A Control @14.5 hrs 4.02 2700611296 Control @16.25 hrs 4.37 29141 11916 Control @18 hrs 4.32 3224714522 pH 6.5 @14.5 hrs 3.08 32144 14400 pH 6.5 @16.25 hrs 3.32 3430113731 pH 6.5 @18 hrs 3.56 36511 15578 pH 7.2 @14.5 hrs 5.17 16447  6258pH 7.2 @16.25 hrs 5.12 17739  6609 pH 7.2 @18 hrs 5.12 21214  7368 pH8.0 @14.5 hrs 4.56 1095   191* pH 8.0 @16.25 hrs 4.38 1451   318* pH 8.0@18 hrs 3.61 1500   281* *Below LOQ at dilution testedConclusions

The highest yields of both Toxin A and Toxin B were produced bymaintaining the pH of the culture at a low pH (i.e., 6.5). The controlculture, which was subject to no pH control also showed significantlymore toxin production than those cultures subjected to a controlled pH7.2 or pH 8.0. Lacking pH control, the pH of the control culturedeclined naturally (typically, declining from a starting pH ofapproximately pH 7.3 to a final pH of approximately 6.3).

SDS-Page gels showed similar bands and intensities for the control andpH 6.5, with the only differences being in the intensity of a band inthe 100 kDa range.

Example 7

This example includes data on the amount of toxin produced whenClostridium difficile is cultured under pH controlled conditions in SYSbasal media having a reduced sodium bicarbonate concentration of 2 g/Land supplemented with D-Sorbitol (12 g/L).

The materials and methods utilized in this experiment were as set out inExample 6, except as noted below.

Materials

SYS media+Sorbitol included 2 g/L NaHCO₃ (reduced from 5 g/L used inExample 6)

Methods

III. Production Bioreactor

-   1. 5×1 L vessels (3^(rd) stage vessels: QB2, QB3, QA1, QA2, QA3)    were prepared in 2 sets (of 2 and 3). The desired temperature and pH    control setpoints were implemented (see Table 6).-   2. Prior to inoculation, bioreactors were sparged with an    appropriate gas for 30 minutes at 300 mL/min (see Table 6).-   3. 3^(rd) stage cultures were incubated at 37° C. with no gassing    for 21 hours.

TABLE 6 Vessel Volume Inoc Temp Stirrer pH NaHCO₃ Degas Sparge CultureNo. (ml) (ml) ° C. (rpm) Control* (g/L) Gas Gas Stage B1 1600  4 37 100None 5 Gas mix Gas mix 1^(st) stage B2 1800 200 37 100 None 5 Gas mixGas mix 2^(nd) stage QA1  900 100 37 100 7.0 2 Gas mix None 3^(rd) stageQA2  900 100 37 100 7.5 2 Gas mix None 3^(rd) stage QA3  900 100 37 100None 2 Gas mix None 3^(rd) stage QB2  900 100 37 100 6.0 2 100% None3^(rd) stage CO₂ QB3  900 100 37 100 6.5 2 100% None 3^(rd) stage CO₂*pH control using 1N HCl, 5N NaOHResults

-   1. The following table shows total cell growth (OD 600 nm) and the    amount of Toxin A produced (ng/ml) and Toxin B produced (ng/ml) in    the cultures subject to the indicated pH control (FIGS. 7A, 7B).

Toxin A Toxin B Sample (Vessel No.) OD@600 nm (ng/ml) (ng/ml) 1^(st)stage 18 h 1.59 N/A N/A 2^(nd) stage 10 h 2.64 N/A N/A pH 6.0 15 h (QB2)1.55 10364 6670 pH 6.5 15 h (QB3) 2.82 26012 16119 pH 7.0 15 h (QA1)4.73 24928 12383 pH 7.5 15 h (QA2) 4.93 7688 2292 Control 15 h (QA3)3.25 30259 20561 pH 6.0 18 h (QB2) 1.49 9047 5785 pH 6.5 18 h (QB3) 3.2133477 20031 pH 7.0 18 h (QA1) 5.39 24702 10588 pH 7.5 18 h (QA2) 4.557694 1882 Control 18 h (QA3) 3.72 46454 22015 pH 6.0 21 h (QB2) 1.7513473 5254 pH 6.5 21 h (QB3) 3.53 38631 17972 pH 7.02 1 h (QA1) 4.8329123 10538 pH 7.5 21 h (QA2) 4.96 7484 1816 Control 21 h (QA3) 4.0141521 20046

-   2. Specific Toxin A productivity produced (ng/ml per OD unit) in the    cultures subject to the indicated pH control is set out in FIG. 7C.    Specific Toxin B productivity produced (ng/ml per OD unit) in the    cultures subject to the indicated pH control is set out in FIG. 7D.    Conclusions

Lowering the sodium bicarbonate to 2 g/L in the SYS+Sorbitol mediumallowed for a lower starting pH with less acid and/or CO₂ sparged. It ispossible to achieve a pH of 6.5 without the addition of acid, bysparging with CO₂.

The uncontrolled pH condition in this experiment had at least equivalenttotal Toxin B production and slightly higher total Toxin A productionthan the pH 6.5 condition. Specific toxin production was similar for theuncontrolled and pH 6.5 conditions.

FIG. 7E depicts a comparison of the results from this experiment andthat set out in Example 6. FIG. 7E shows total toxin concentration at 18h for various conditions over the 2 experiments. A clear drop-off intoxin production is seen in cultures at pH 6.5 to pH 6.0 and a moregradual decline in toxin production in the higher pH conditions. Theoptimal pH is slightly higher than 6.5.

Example 8

This example includes data on the effect of different concentrations ofsodium bicarbonate (i.e., 0 g/L, 2 g/L, and 5 g/L) and carbon dioxidesparing on the pH of SYS basal media supplemented with D-Sorbitol (12g/L).

Materials

The following materials and equipment were used in this example.

The Biostat Q Plus Fermentation System (Sartorius) was used for thefirst, second, and third stage cultures, using three 1 L fermenters(Sartorius). The composition of the SYS media supplemented withD-Sorbitol (SYS media+Sorbitol) was as described in Example 6, exceptthat no NaHC0₃ was added to the initial 4 L batch prepared.

Methods

The following methods were used to test changes in pH.

-   1. 3 pH probes were calibrated on the Biostat Q Plus system-   2. 4 L of SYS media with sorbitol was made without sodium    bicarbonate and 1 L was added to a 1 L fermenter.-   3. 6 g of sodium bicarb was added to the remaining 3 L of media for    a bicarb concentration of 2 g/L. 1 L of the media was added to a 1 L    fermenter.-   4. 6 g of sodium bicarb was added to the remaining 2 L of media for    a bicarb concentration of 5 g/L. 1 L of the media was added to a 1 L    fermenter.-   5. All fermenters were mixed at 100 rpm and the pH probes were    installed-   6. 100% CO₂ was sparged at 500 ml/min and the data acquisition    software was started to generate pH curves-   7. After ˜3.5 hours, 5 ml of 5N HCl was added to each fermenter.    Results-   1. The following table shows the pH changes noted using different    concentrations of sodium bicarbonate.

Lowest pH with pH with 5 ml 5N Condition Starting pH CO₂ sparging HCladded 0 g/L Bicarb 7.15 6.18 5.72 2 g/L Bicarb 7.15 6.28 5.94 5 g/LBicarb 7.12 6.4 6.1Conclusions

A lower final pH can be achieved in SYS medium with a lower sodiumbicarbonate concentration when gassing with CO₂. Using 2 g/L bicarb canlower the pH by 0.12 units with CO₂ sparge alone compared to 5 g/L.

Example 9

This example includes data on the amount of toxin produced whenClostridium difficile is cultured in SYS basal media supplemented withD-Sorbitol (12 g/L) with different concentrations of sodium bicarbonate(i.e., 0 g/L, 2 g/L, and 5 g/L) and spared with carbon dioxide or ananaerobic gas mix (80% N₂/10% CO₂/10% H₂).

The materials and methods utilized in this experiment were as set out inExample 6, except as noted below.

Materials

The composition of the SYS media for the first stage seed cultures wasas described in Example 6. For the third stage cultures, an SYS mediasupplemented with Sorbitol was prepared having the following compositionand using RODI-water:

SYS media + Sorbitol Formulation Component Manufacturer/Lot # (g/L)KH₂PO₄ J T Baker/E29H22 0.9 Na₂PO₄ J T Baker/A12145 5 NaHCO₃ J TBaker/A13668 0 Soy Peptone A3 SC Organotechnie/19685 30 Yeast Extract BDBacto Part#21270/Lot 20 #8352570 D-Sorbitol (70%) Spectrum/WJ1030 17.1mlTwo separate batches of SYS media+Sorbitol culture media were alsoprepared having the same composition but with a different concentrationof NaHCO₃ (i.e., one with NaHCO₃ 2 g/L and one with 5 g/L NaHCO₃).MethodsIII. Production Bioreactor

-   1. 5×1 L vessels (3^(rd) stage vessels: QB2, QB3, QA1, QA2, QA3)    were prepared in 2 sets (of 2 and 3). The desired temperature and pH    control setpoints were implemented (see Table 7).-   2. Prior to inoculation, bioreactors were sparged with an    appropriate gas for 30 minutes at 300 mL/min (see Table 7).-   3. 3^(rd) stage cultures were incubated at 37° C. with no gassing    for 21 hours.

TABLE 7 Vessel Volume Inoc Temp Stirrer pH NaHCO₃ Degas Sparge CultureNo. (ml) (ml) ° C. (rpm) Control* (g/L) Gas Gas Stage B1 1600  4 37 100None 5 Gas mix Gas mix 1^(st) stage B2 1800 200 37 100 None 5 Gas mixGas mix 2^(nd) stage QA1  900 100 37 100 None 0 Gas mix None 3^(rd)stage QA2  900 100 37 100 None 2 Gas mix None 3^(rd) stage QA3  900 10037 100 None 5 Gas mix None 3^(rd) stage QB2  900 100 37 100 6.5 2 100%None 3^(rd) stage CO₂ QB3  900 100 37 100 6.5 2 Gas mix None 3^(rd)stage *pH control using 1N HCl, 5N NaOHResults

-   1. The following table shows total cell growth (OD 600 nm) and the    amount of Toxin A produced (ng/ml) and Toxin B produced (ng/ml) in    the cultures subject to the indicated pH control (FIGS. 8A, 8B).

Toxin A Toxin B Sample OD@600 nm (ng/ml) (ng/ml) 1^(st) stage 18 h 0.76N/A N/A 2^(nd) stage 10 h 2.27 N/A N/A QB2- pH 6.5 CO₂ 16 h 2.82 1851910519 QB3- pH 6.5 gas mix 16 h 2.71 22109 11051 QA1- 0 g/L bicarb 16 h2.73 22898 10217 QA2- 2 g/L bicarb 16 h 3.22 29048 15099 QA3- 5 g/Lbicarb 16 h 4.02 25579 12087 QB2- pH 6.5 CO2 18 h 2.98 22820 13695 QB3-pH 6.5 gas mix 18 h 2.97 25185 14463 QA1- 0 g/L bicarb 18 h 3.15 2568811576 QA2- 2 g/L bicarb 18 h 3.5 34927 18500 QA3- 5 g/L bicarb 18 h 3.8628656 15256

-   2. The 1st stage cell growth in this experiment was lower than    typically seen in this experiment. There was not a significant    difference in the growth or toxin production of the fermentations    controlled at pH 6.5 with either CO₂ or anaerobic gas mix sparging.    A sodium bicarbonate concentration of 2 g/L sodium bicarbonate    provided a higher specific and total toxin A and B productivity    compared to concentrations of 0 g/L and 5 g/L.    Conclusions

The use of CO₂ for degassing the media is an option when controlling pHat 6.5 because of the comparable toxin yields to the anaerobic gas mixdegassed fermentation.

Example 10

This example includes data on the amount of toxin produced whenClostridium difficile is cultured under a range of temperatures (37-41°C. with a midpoint of 39° C.) and a range of pH (6.35 to 6.65 with amidpoint of 6.5) in SYS basal media supplemented with D-Sorbitol (12g/L).

The materials and methods utilized in this experiment were as set out inExample 6, except as noted below.

Materials

The composition of the SYS media for the first stage seed cultures wasas described in Example 6. For the third stage cultures, an SYS mediasupplemented with Sorbitol was prepared having the following compositionand using RODI-water:

SYS media + Sorbitol Formulation Component Manufacturer/Part #/Lot #(g/L) KH₂PO₄ J T Baker/3248-07/Y48478 0.9 Na₂PO₄ J TBaker/3827-01/B08143 5 NaHCO₃ J T Baker/3509-05/E05589 2 Soy Peptone A3SC Organotechnie/130-127- 30 00/19685 Yeast Extract BDBacto/212720/8352570 20 D-Sorbitol (70%) Spectrum/S0220/WJ1030 17.1 mlMethodsIII. Production Bioreactor

-   1. 6×1 L vessels (3^(rd) stage vessels: QB1, QB2, QB3, QA1, QA2,    QA3) were prepared in 2 sets (of 2 and 3). The desired temperature    and pH control setpoints were implemented (see Table 8).-   2. Prior to inoculation, bioreactors were sparged with an    appropriate gas for 30 minutes at 300 mL/min (see Table 8).-   3. 3^(rd) stage cultures were incubated at the applicable    temperature with no gassing for 21 hours.

TABLE 8 Vessel Volume Inoc Temp Stirrer pH NaHCO₃ Degas Sparge CultureNo. (ml) (ml) ° C. (rpm) Control (g/L) Gas Gas Gas B1 1600  4 37 100None 5 Gas mix Gas mix 1^(st) stage B2 1800 200 37 100 None 5 Gas mixGas mix 2^(nd) stage QA1  900 100 39 100 None 2 Gas mix none 3^(rd)stage QA2  900 100 39 100 6.5  2 Gas mix none 3^(rd) stage QA3  900 10037 100 6.35 2 Gas mix none 3^(rd) stage QB1  900 100 37 100 6.65 2 Gasmix none 3^(rd) stage QB2  900 100 41 100 6.35 2 Gas mix none 3^(rd)stage QB3  900 100 41 100 6.65 2 Gas mix none 3^(rd) stage Gas mixutilized was 80% N₂/10% CO₂/10% H₂Results

-   1. The following table shows total cell growth (OD 600 nm) and the    amount of Toxin A produced (ng/ml) and Toxin B produced (ng/ml) in    the cultures subject to the indicated temperature and the indicated    pH control (FIGS. 9A, 9B).

Toxin A Toxin B Sample OD@600 nm (ng/ml) (ng/ml) 1^(st) stage 18 h 0.84N/A N/A 2^(nd) stage 10 h 1.92 N/A N/A QA1 39° C. uncontrolled 18 h 3.2329892 18321 QA2 39° C. pH 6.5 18 h 3.24 32565 18445 QA3 37° C. pH 6.3518 h 2.83 21827 9173 QB1 37° C. pH 6.65 18 h 3.32 33149 18508 QB2 41° C.pH 6.35 18 h 2.43 21537 13522 QB3 41° C. pH 6.65 18 h 3.14 25924 16784QA1 39° C. uncontrolled 21 h 3.26 27314 13886 QA2 39° C. pH 6.5 21 h3.20 30509 13658 QA3 37° C. pH 6.35 21 h 3.04 34317 16935 QB1 37° C. pH6.65 21 h 3.41 24851 14450 QB2 41° C. pH 6.35 21 h 2.90 21176 16561 QB341° C. pH 6.65 21 h 2.95 28002 20790

-   2. Cell growth was higher in lower temperature and higher pH    conditions. Toxin A production was higher in the low temperature    (37° C.) and low pH (6.35) conditions. Toxin B production was higher    in the high temperature (41° C.) and high pH (6.65) conditions.    Lower toxin A yields were seen in high temperature and low pH    conditions and lower toxin B yields were seen in low temperature and    high pH conditions.    Conclusions

Optimal conditions for production of Toxin A and B are different. SinceToxin B availability is a limiting factor for the production of avaccine product comprising Toxoids A and B (e.g., in a ratio of 3:2),conditions which favor Toxin B production may be preferred. Highertemperature (41° C.) and higher controlled pH (6.65) are the bestconditions for Toxin B production. While these conditions are not themost optimal for Toxin A yields, Toxin A is produced and at a levelabove other conditions.

Example 11

This example includes data on the amount of toxin produced whenClostridium difficile is cultured in SYS basal media supplemented withD-Sorbitol (12 g/L) under a controlled a pH 6.5, and at varioustemperatures (33, 35, 37, 39, 41, 43° C.) and 2 g/L sodium bicarbonate.

The materials and methods utilized in this experiment were as set out inExample 6, except as noted below.

Materials

The composition of the SYS media for the first stage seed cultures wasas described in Example 6. For the third stage cultures, an SYS mediasupplemented with Sorbitol was prepared having the following compositionand using RODI-water:

SYS media + Sorbitol Formulation Component Manufacturer/Part #/Lot #(g/L) KH₂PO₄ J T Baker/3248-07/E29H22 0.9 Na₂PO₄ J TBaker/3827-01/B08143 5 NaHCO₃ J T Baker/3509-05/E05589 2 Soy Peptone A3SC Organotechnie/130-127- 30 00/102630 Yeast Extract BDBacto/212720/8352570 20 D-Sorbitol (70%) Spectrum/S0220/WJ1030 17.1 mlMethodsIII. Production Bioreactor

-   1. 6×1 L vessels (3^(rd) stage vessels: QA1 to QA3, QB1 to QB3) were    prepared in 2 sets (of 2 and 3). The desired temperature and pH    control setpoints were implemented (see Table 9).-   2. Prior to inoculation, bioreactors were sparged with an    appropriate gas for 30 minutes at 300 mL/min (see Table 9).-   3. 3^(rd) stage cultures were incubated at the applicable    temperature with no gassing for 21 hours.

TABLE 9 Sod- ium Vess- Vol- Bi- Cul- el ume Ionc. Temp pH carb Spargeture No. (ml) (ml) (° C.) Control (g/L) Degas Gas Gas Gas B1 1600  4 37None 5 Gas mix Gas mix 1^(st) stage B2 1800 200 37 None 5 Gas mix Gasmix 2^(nd) stage QA1  900 100 33 6.5  2 100% CO₂ None 3^(rd) stage QA2 900 100 35 6.5* 2 100% CO₂ None 3^(rd) stage QA3  900 100 37 6.5  2100% CO₂ None 3^(rd) stage QB1  900 100 39 6.5  2 100% CO₂ None 3^(rd)stage QB2  900 100 41 6.5  2 100% CO₂ None 3^(rd) stage QB3  900 100 436.5  2 100% CO₂ None 3^(rd) stage *pH control for QA2 was not activateddue to a problem with the base tubing. Gas mix utilized was 80% N₂/10%CO₂/10% H₂Results

-   1. The following table shows total cell growth (OD 600 nm) and the    amount of Toxin A produced (ng/ml) and Toxin B produced (ng/ml) in    the cultures subject to the indicated temperature and the indicated    pH control (FIGS. 10A, 10B, 10C, 10D).

Toxin A Toxin B Sample OD@600 nm (ng/ml) (ng/ml) 1^(st) stage 18 h 1.31N/A N/A 2^(nd) stage 10 h 1.90 N/A N/A QA1 19 h 2.40 10956 5853 QA2 19 h2.42 15409 9487 QA3 19 h 2.95 19723 13736 QB1 19 h 2.12 18425 16929 QB219 h 2.53 18465 18184 QB3 19 h 1.71 9981 9110 QA1 22 h 2.57 12200 8218QA2 22 h 2.73 16895 10681 QA3 22 h 2.74 29124 22679 QB1 22 h 2.20 1768616658 QB2 22 h 2.46 18730 22104 QB3 22 h 1.76 10160 9091

-   2. Cell growth decreases at temperatures higher than 37° C. Toxin A    production is highest and similar within the range of 37-41° C.    Toxin B yield increases almost linearly with increasing temperature    from 37-41° C.    Conclusions

Culturing C. difficile at 37-41° C. is optimal for both Toxin A and Bproduction. Culturing C. difficile at temperatures at the higher end ofthe 37-41° C. range favors increased Toxin B production.

Example 12

This example includes data on the amount of toxin produced whenClostridium difficile is cultured in SYS basal media supplemented withD-Sorbitol (12 g/L) using different inoculum concentrations (1%, 5%, and10% of initial bioreactor volume) and under different pH conditions(controlled pH 6.5 and controlled at pH 6.5 with base-only).

The materials and methods utilized in this experiment were as set out inExample 6, except as noted below.

Materials

The composition of the SYS media for the first stage seed cultures wasas described in Example 6. For the third stage cultures, an SYS mediasupplemented with Sorbitol was prepared having the following compositionand using RODI-water:

SYS media + Sorbitol Formulation Component Manufacturer/Part #/Lot #(g/L) KH₂PO₄ J T Baker/3248-07/Y48478 0.9 Na₂PO₄ J TBaker/3827-01/B08143 5 NaHCO₃ J T Baker/3509-05/E05589 2 Soy Peptone A3SC Organotechnie/130-127-00/18 30 Yeast Extract BD Bacto/212720/835257020 D-Sorbitol (70%) Spectrum/S0220/WJ1030 17.1 ml (12 g/L)MethodsIII. Production Bioreactor

-   1. 6×1 L vessels (3^(rd) stage vessels: QA1 to QA3, QB1 to QB3) were    prepared in 2 sets (of 2 and 3). The desired temperature and pH    setpoints were implemented (see Table 11). For vessels QA1, QA2, and    QA3, pH was set at 6.5 for control with base-only (5N NaOH).    Base-only control involves the addition of base to the culture to    adjust the culture pH to pH 6.5 in the event the culture pH becomes    lower than 6.5. Under such control, the pH of the culture naturally    decreases from the initial media pH (approximately pH 7.4) to pH    6.5.-   2. Prior to inoculation, bioreactors were sparged with the    applicable gas for 30 minutes at 300 mL/min (see Table 10) and then    an overlay of nitrogen gas was added to the applicable vessels.-   3. 3^(rd) stage cultures were incubated at the applicable    temperature for 24 hours.

TABLE 10 Sod- ium Vess- Vol- Bi- Sparge Cul- el ume Inoc Temp pH carbDegas Gas ture No. (ml) (ml) (° C.) Control (g/L) Gas (overlay) Stage B11600  4 37 none 5 Gas mix Gas mix 1^(st) stage B2 1800 200 37 none 5 Gasmix Gas mix 2^(nd) stage QA1  900  10 37 Low end 2 Nitrogen (Nitrogen)3^(rd) 6.5 stage QA2  900  50 37 Low end 2 Nitrogen (Nitrogen) 3^(rd)6.5 stage QA3  900 100 37 Low end 2 Nitrogen (Nitrogen) 3^(rd) 6.5 stageQB1  900  10 37 6.5 2 CO₂ (Nitrogen) 3^(rd) stage QB2  900  50 37 6.5 2CO₂ (Nitrogen) 3^(rd) stage QB3  900 100 37 6.5 2 CO₂ (Nitrogen) 3^(rd)stageResults

The following table shows total cell growth (OD 600 nm) and the amountof Toxin A produced (ng/ml) and Toxin B produced (ng/ml) in the culturessubject to the indicated temperature and the indicated pH control (FIG.11).

Cell Growth by OD600 Toxin A Concentration By Toxin B Concentration byMeasurement ELISA ELISA Sample 0 h 16 h 20 h 24 h 16 h 20 h 24 h 16 h 20h 24 h QA1 low end  0.013 4.24 4.75 4.46 12840 23135 24056 7006.115679.21 16010.07 pH 6.5, 1% inoc QA2 low end  0.089 4.71 4.64 4.6217831 24387 26873  9599.37 13386.5  17642.25 pH 6.5, 5% inoc QA3 low end 0.182 4.49 4.76 4.39 19978 34845 29240 12300.11 18254.64 16550.53 pH6.5, 10% inoc QB1 pH 6.5, −0.016 3.11 4.08 4.25 13757 28501 34720 9002.91 16828.93 25691.37 1% inoc QB2 pH 6.5,  0.062 3.22 4.29 4.5919815 33186 34216 11500.96 21603.89 21967.57 5% inoc QB3 pH 6.5,  0.1533.56 4.5  4.46 26469 35068 44541 16546.46 21293.34 34246.13 10% inoc

In this experiment, a 10 L vessel (Sartorius) was also utilized. Thevessel was autoclaved and connected to the Biostat system and thefollowing conditions were set: 37° C. and agitation (stirring) at 100rpm. Culture pH was not controlled. The vessel was filled with 9 L ofthe SYS media also utilized in filling the 1 L fermenters (i.e., SYSmedia with 12 g/L sorbitol and 2 g/L Na₂HCO₃). The vessel was thende-gassed using Nitrogen gas and inoculated with 1 L of the SeedBioreactor 2 culture. Toxin production and cell growth (OD) was measuredfollowing an 18 hour incubation: Toxin A (24533 ng/ml); Toxin B (14837ng/ml); 2.94 OD(600 nm).

A 10 L vessel was also included in two of the experiments set out above(i.e., Examples 10 and 11) and was prepared, inoculated, and culturedsimilarly (except de-gassing was done with gas mix 80% N₂/10% CO₂/10% H₂and agitation was set at 75 rpm). The measured toxin production and cellgrowth following an 18 hour incubation was as follows: in Example 10,Toxin A (29605 ng/ml); Toxin B (10732 ng/ml); 2.95 OD(600 nm); inExample 11, Toxin A (25681 ng/ml); Toxin B (24898 ng/ml); 3.17 OD (600nm). In a separate experiment, toxin production and cell growth in a 10L culture with SYS media (with 12 g/L sorbitol and 2 g/L Na₂HCO₃) undersimilar conditions (i.e., a 10% inoculum concentration, culturetemperature of 37° C. and 50 rpm agitation) was similar: Toxin A (21090ng/ml); Toxin B (12228 ng/ml) and 3.02 OD (600 nm).

Conclusions

Similar toxin yields may be achieved by using inoculum rates lower than10% although the culture duration may need to be increased. Inoculationsof 1% and 5% achieved toxin yields >30 μg/ml for toxin A and >15 μg/mlof toxin B after 20 hours.

Other Embodiments

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure that come within known or customary practice withinthe art to which the invention pertains and may be applied to theessential features hereinbefore set forth.

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindependent publication or patent application was specifically andindividually indicated to be incorporated by reference in theirentirety.

What is claimed is:
 1. A method for obtaining one or more Clostridiumdifficile (C. difficilel) toxins comprising the steps of: (a) preparingan aqueous growth medium comprising soy peptone and D-sorbitol; (b)inoculating the medium with a C. difficile bacterium; (c) culturing theinoculated medium under conditions which facilitate growth of bacteriumand toxin production; and (d) isolating the one or more C. difficiletoxins from the growth medium.
 2. The method of claim 1, wherein: (a) instep (a) the growth medium comprises: (i) between 10 and 20 g/L soypeptone; (ii) between 10 and 30 g/I yeast extract; between 2 and 5 g/LNaHCO₃; between 1 and 10 g/L sodium phosphate, dibasic; between 1 and 10g/L potassium phosphate, monobasic; and between 6 and 20 g/L D-sorbitol;(b) in step (c) the inoculated medium is at pH of between 6.35 and 6.65during culturing; (c) in step (c) the culturing of the inoculated mediumtakes place at 37° C. to 41° C.; (d) the culturing of the inoculatedmedia is carried out under anaerobic conditions; or (e) in step (b) themedium is inoculated with an aqueous C. difficile culture.
 3. The methodof claim 1, further comprising the step of detoxifying the isolated oneor more C. difficile toxins to prepare one or more toxoids.
 4. Themethod of claim 3, wherein: (a) steps (b) and (c) are repeated more thanonce, with inoculation into fresh growth medium in each repeat; (b)wherein step (c) takes place at 37° C. to 41° C.; (c) step (d)comprises: removing from the growth medium viable C. difficile organismsand spores, separating the one or more toxins from the growth media, andpurifying the one or more toxins; or (d) step (e) comprises reacting theone or more toxins with the addition of formaldehyde.
 5. The method ofclaim 1, wherein: (a) the growth medium further comprises: at least oneadditive selected from the group consisting of chromium trioxide,clindamycin, ascorbic acid, butyric acid, D(+)xylose, sucrose, and acombination of azaserine, adenosine, and biotin.
 6. The method of claim5, wherein the growth medium comprises: (a) at least two of saidadditives; (b) D-sorbitol at a concentration between 6 g/L and 20 g/L;(c) D-sorbitol at a concentration of 12 g/L; (d) chromium trioxide at aconcentration between 40 and 60 mg/L; (e) chromium trioxide at aconcentration of 50 mg/L; (f) clindamycin at a concentration between 0.4and 0.6 mg/L; (g) clindamycin at a concentration of 0.5 mg/L; (h)ascorbic acid at a concentration between 2.5 g/L and 10 g/L; (i)ascorbic acid at a concentration selected from 2.5 g/L and 10 g/L; (j)butyric acid at a concentration between 30 mM and 60 mM; (k) butyricacid at a concentration selected from 30 mM and 60 mM; (I) D(+)xylose ata concentration between 6 and 10 g/L; (m) D(+)xylose at a concentrationof 6 g/L; (n) adenosine at a concentration of between 0.8 and 1.2 mM,biotin at a concentration of between 40 and 60 nM, and azaserine at aconcentration between 15 and 50 μM; or (o) adenosine at a concentrationof 1 mM, biotin at a concentration of 50 nM, and azaserine at aconcentration of 50 μM.
 7. The method of claim 6, wherein the growthmedium comprises soy peptone, yeast extract, KH₂PO₄, Na₂HPO₄, andNaHCO₃, and wherein the culture is at a pH of between 6.35 and 7.45. 8.A method of enhancing the production from a C. difficile culture ofToxin B relative to the production of Toxin A comprising the steps of:(a) preparing an aqueous growth medium comprising soy peptone andD-sorbitol; (b) inoculating the medium with a C. difficile bacterium;and (c) culturing the inoculated medium at 37° C. to 41° C.
 9. Themethod of claim 8, wherein: (a) step (c) takes place at 37° C. and at apH of pH 6.5; (b) the pH of step (c) is controlled at a pH between pH6.35 to pH 6.65; or (c) the aqueous growth medium of step (a) furthercomprises between 10 and 20 g/L soy peptone; and between 6 and 20 g/LD-sorbitol.
 10. The method of claim 8, wherein: (a) the production ofToxin A relative to Toxin B is less than 3:1; (b) the production ofToxin A relative to Toxin B is less than 2:1; or (c) the production ofToxin A relative to Toxin B is equal to or less than 1.5:1.